Polarized fluorescence spectroscopy of oriented isolated spinach Photosystem I particles

The fluorescence anisotropy of Photosystem I (PS I) particles, isolated from spinach chloroplasts and containing approximately 200 chlorophyll molecules per reaction center, is investigated at low temperatures. The particles are oriented by squeezing in polyacrylamid gels with different macroscopic deformation parameters. Fluorescence anisotropy is measured upon steady state excitation with a laser line at 632.8 nm. A formula for the fluorescence anisotropy in oriented Photosystem I particles is applied for a different polarization of the linearly polarized exciting light. Our calculations are based on the consideration of the Photosystem I complex as a triple-chromophore complex: the absorbing chlorophyll molecules (chl), belonging to the light-harvesting complex of PS I (LHC), and two fluorophores, emitting at 720 nm (F720) and at 735 nm (F735), respectively. Using polarized fluorescence spectroscopy with a different polarization of the linearly polarized exciting light, the experimental dependence of the fluorescence anisotropy on this polarization is obtained. Based on this dependence and applying the derived formula, as a first approximation, both the orientation of the photosynthetic pigments with respect to the membrane and their mutual orientation are determined in PS I particles. As the most probable average orientational angles in PS I particles, we obtained the values 35°÷ 50°, 50°÷ 60°, and 65°÷ 67° for the absorbing dipoles of chl and for the emission dipoles of F720 and F735, respectively, with the normal of the plane of the membrane. For their mutual orientation, the following limits are determined: 10°÷ 20°, 40 ± 2°, 20°÷ 30° for the angles between chl and F720, chl and F735; and F720 and F735, correspondingly. Of course, the values of the angles estimated as a result of our study are an average value of all angles of the excited transitions and must be considered as their first approximation valid for the idealized case when all PS I particles are oriented in gel. This revised version was published online in June 2006 with corrections to the Cover Date.     

Biosorption of binary mixtures of copper and cobalt by Penicillium brevicompactum

This work reports on a study of the biosorption of copper and cobalt, both singly and in combination (in equimolar concentrations), by the resting cells of Penicillium brevicompactum. Equilibrium batch sorption studies were carried out at 30 degrees C and pH 5.0 for a contact time of 1 hour to guarantee that equilibrium was reached. The equilibrium data were analyzed using the Langmuir and Freundlich isotherms. The adsorption of binary mixtures of heavy metal solutions on the fungal biomass was found to be of competitive type where the adsorption capacity for any single metal decreased in the presence of the other. The cobalt ions showed a higher affinity for Penicillium brevicompactum than the copper ions.     

Insights into pathological mechanisms and interventions revealed by analyzing a mathematical model for cone metabolism

This work analyzes a mathematical model for the metabolic dynamics of a cone photoreceptor, which is the first model to account for energy generation from fatty acids oxidation of shed photoreceptor outer segments (POS). Multiple parameter bifurcation analysis shows that joint variations in external glucose, the efficiency of glucose transporter 1 (GLUT1), lipid utilization for POS renewal, and oxidation of fatty acids affect the cone’s metabolic vitality and its capability to adapt under glucose-deficient conditions. The analysis further reveals that when glucose is scarce, cone viability cannot be sustained by only fueling energy production in the mitochondria, but it also requires supporting anabolic processes to create lipids necessary for cell maintenance and repair. In silico experiments are used to investigate how the duration of glucose deprivation impacts the cell without and with a potential GLUT1 or oxidation of fatty acids intervention as well as a dual intervention. The results show that for prolonged duration of glucose deprivation, the cone metabolic system does not recover with higher oxidation of fatty acids and requires greater effectiveness of GLUT1 to recover. Finally, time-varying global sensitivity analysis (GSA) is applied to assess the sensitivity of the model outputs of interest to changes and uncertainty in the parameters at specific times. The results reveal a critical temporal window where there would be more flexibility for interventions to rescue a cone cell from the detrimental consequences of glucose shortage.     

Exploring the robustness of macrophyte-based classification methods to assess the ecological status of coastal and transitional ecosystems under the Water Framework Directive

Identifying and quantifying the factors that contribute to the potential misclassification of the ecological status of water bodies is a major challenge of the Water Framework Directive (WFD). The present study compiles extensive biomonitoring data from a range of macrophyte-based classification methods developed by several European countries. The data reflect spatial and temporal variation as well as inter-observer variation. Uncertainty analysis identified that factors related to the spatial scale of sampling generally contributed most to the uncertainty in classifying water bodies to their ecological status, reflecting the high horizontal and depth-related heterogeneity displayed by macrophyte communities. In contrast, the uncertainty associated with temporal variation was low. In addition, inter-observer variation, where assessed, did not contribute much to overall uncertainty, indicating that these methods are easily transferable and insensitive to observer error. The study, therefore, suggests that macrophyte-based sampling schemes should prioritize large spatial replication over temporal replication to maximize the effectiveness and reliability of water body classification within the WFD. We encourage conducting similar uncertainty analyses for new/additional ecological indicators to optimize sampling schemes and improve the reliability of classification of ecological status.     

Diversity of European seagrass indicators: patterns within and across regions

Seagrasses are key components of coastal marine ecosystems and many monitoring programmes worldwide assess seagrass health and apply seagrasses as indicators of environmental status. This study aims at identifying the diversity and characteristics of seagrass indicators in use within and across European ecoregions in order to provide an overview of seagrass monitoring effort in Europe. We identified 49 seagrass indicators used in 42 monitoring programmes and including a total of 51 metrics. The seagrass metrics represented 6 broad categories covering different seagrass organizational levels and spatial scales. The large diversity is particularly striking considering that the pan-European Water Framework Directive sets common demands for the presence and abundance of seagrasses and related disturbance-sensitive species. The diversity of indicators reduces the possibility to provide pan-European overviews of the status of seagrass ecosystems. The diversity can be partially justified by differences in species, differences in habitat conditions and associated communities but also seems to be determined by tradition. Within each European region, we strongly encourage the evaluation of seagrass indicator–pressure responses and quantification of the uncertainty of classification associated to the indicator in order to identify the most effective seagrass indicators for assessing ecological quality of coastal and transitional water bodies.     

Cell cycle specific plasmid DNA replication in the nuclear extract of Saccharomyces cerevisiae: modulation by replication protein A and proliferating cell nuclear antigen

Plasmid DNA replication in nuclear extracts of Saccharomyces cerevisiae in vitro has been shown to be S-phase specific, similar to that observed in vivo. We report here a reconstituted in vitro system with partially purified replication proteins, purified replication protein A (RPA), and recombinant proliferating cell nuclear antigen (PCNA). Nuclear extracts from S-phase, G(1)-phase, and unsynchronized yeast cells were fractionated by phosphocellulose chromatography. Protein fraction (polymerase fraction) enriched with replication proteins, including DNA polymerases (alpha, delta, etc.), was isolated, which was not capable of in vitro replication of supercoiled plasmid DNA. However, when purified yeast RPA and recombinant PCNA together were added to the polymerase fraction obtained from S-phase synchronized cells, in vitro plasmid DNA replication was restored. In vitro plasmid DNA replication with polymerase fractions from unsynchronized and G(1)-phase cells could not be reconstituted upon addition of purified RPA and PCNA. RPA and PCNA isolated from various phases of the cell cycle complemented the S-phase polymerase pool to the same extent. Reconstituted systems with the S-phase polymerase pool, complemented with either the RPA- and PCNA-containing fraction or purified RPA and recombinant PCNA together, were able to produce replication intermediates (ranging in size from 50 to 1500 bp) similar to that observed with the S-phase nuclear extract. Results presented here demonstrate that both RPA and PCNA are cell cycle-independent in their ability to stimulate in vitro plasmid DNA replication, whereas replication factors in the polymerase fractions are strictly S-phase dependent.     

Heat- and light-induced reorganizations in the phycobilisome antenna of Synechocystis sp. PCC 6803. Thermo-optic effect

By using absorption and fluorescence spectroscopy, we compared the effects of heat and light treatments on the phycobilisome (PBS) antenna of Synechocystis sp. PCC 6803 cells. Fluorescence emission spectra obtained upon exciting predominantly PBS, recorded at 25 degrees C and 77 K, revealed characteristic changes upon heat treatment of the cells. A 5-min incubation at 50 degrees C, which completely inactivated the activity of photosystem II, led to a small but statistically significant decrease in the F(680)/F(655) fluorescence intensity ratio. In contrast, heat treatment at 60 degrees C resulted in a much larger decrease in the same ratio and was accompanied by a blue-shift of the main PBS emission band at around 655 nm (F(655)), indicating an energetic decoupling of PBS from chlorophylls and reorganizations in its internal structure. (Upon exciting PBS, F(680) originates from photosystem II and from the terminal emitter of PBS.). Very similar changes were obtained upon exposing the cells to high light (600-7500 micromol photons m(-2) s(-1)) for different time periods (10 min to 3 h). In cells with heat-inactivated photosystem II, the variations caused by light treatment could clearly be assigned to a similar energetic decoupling of the PBS from the membrane and internal reorganizations as induced at around 60 degrees C. These data can be explained within the frameworks of thermo-optic mechanism [Cseh et al. 2000, Biochemistry 39, 15250]: in high light the heat packages originating from dissipation might lead to elementary structural changes in the close vicinity of dissipation in heat-sensitive structural elements, e.g. around the site where PBS is anchored to the membrane. This, in turn, brings about a diminishment in the energy supply from PBS to the photosystems and reorganization in the molecular architecture of PBS.     

Evaluation of antimicrobial resistance among <Emphasis Type="Italic">Salmonella</Emphasis> and <Emphasis Type="Italic">Shigella</Emphasis> isolates in the University Hospital “St. George,” Plovdiv,Bulgaria

The aim of this work is to study the epidemiology and antimicrobial resistance to the most commonly used antibiotics for the treatment of acute gastroenteritis caused by Salmonella and Shigella at the largest Bulgarian hospital—University Hospital “St. George,” Plovdiv—for the period 2009–2013. Two hundred ninety strains were in vitro tested for resistance to 15 antimicrobial agents. The presence of extended-spectrum beta-lactamases (ESBLs) was demonstrated by a variety of specialized tests. For comparison, a collection of 28 strains submitted by the National Reference Laboratory (NRL) “Enteric Infections” at the National Center of Infectious and Parasitic Diseases (NCIPD), Sofia, was also tested for the production of ESBLs. In isolates, phenotypically demonstrated as ESBL producers, polymerase chain reaction (PCR) detection of the genes bla-CTX-M, bla-SHV, and bla-TEM was performed. Among the 290 tested isolates, only two— Salmonella serotype Livingstone and Shigella flexneri—were phenotypically proven to be ESBL producers. Only 4 strains from the collection of 28, submitted from the NRL “Intestinal Infections” in NCIPD, Sofia, were phenotypically confirmed as ESBL producers. The presence of the bla-CTX-M gene was detected in all of the tested strains (4 from NRL, NCIPD, Sofia, and 2 from the University Hospital St. George, Plovdiv), the bla-SHV gene only in strain S. Livingstone from Plovdiv, and the bla-TEM gene in two from Sofia and one (again S. Livingstone) from Plovdiv. In conclusion, Salmonella and Shigella isolates from patients hospitalized at the University Hospital St. George, Plovdiv, with acute gastroenteritis demonstrate good susceptibility to the most commonly used antibiotic agents, including azithromycin.     

Peroxidase and IAA-oxidase in crude and partially purified enzyme extracts of growing and differentiating root cells

Crude enzyme extracts from the zones of division, elongation and differentiation of cells of primary maize (Zea mays) root show peroxidase activity but lack IAA-oxidase activity. After partial purification of the extracts by gel filtration on Sephadex G-25, the specific peroxidase activity increases almost twice and a high IAA-oxidase activity appears. The partial purification of the enzyme extracts does not change the electrophoretic pattern of the peroxidase isoenzymes, but significantly improves the separation and the visualization of isoenzymes with IAA-oxidase activity. The data obtained were interpreted in connection with the different modifying effect of the low molecular compounds (mainly phenolics) on the activity and the isoenzyme patterns of the peroxidase and the IAA-oxidase.     

Heat- and light-induced reorganizations in the phycobilisome antenna of Synechocystis sp. PCC 6803. Thermo-optic effect

By using absorption and fluorescence spectroscopy, we compared the effects of heat and light treatments on the phycobilisome (PBS) antenna of Synechocystis sp. PCC 6803 cells. Fluorescence emission spectra obtained upon exciting predominantly PBS, recorded at 25 °C and 77 K, revealed characteristic changes upon heat treatment of the cells. A 5-min incubation at 50 °C, which completely inactivated the activity of photosystem II, led to a small but statistically significant decrease in the F₆₈₀/F₆₅₅ fluorescence intensity ratio. In contrast, heat treatment at 60 °C resulted in a much larger decrease in the same ratio and was accompanied by a blue-shift of the main PBS emission band at around 655 nm (F₆₅₅), indicating an energetic decoupling of PBS from chlorophylls and reorganizations in its internal structure. (Upon exciting PBS, F₆₈₀ originates from photosystem II and from the terminal emitter of PBS.). Very similar changes were obtained upon exposing the cells to high light (600-7500 μmol photons m⁻² s⁻¹) for different time periods (10 min to 3 h). In cells with heat-inactivated photosystem II, the variations caused by light treatment could clearly be assigned to a similar energetic decoupling of the PBS from the membrane and internal reorganizations as induced at around 60 °C. These data can be explained within the frameworks of thermo-optic mechanism [Cseh et al. 2000, Biochemistry 39, 15250]: in high light the heat packages originating from dissipation might lead to elementary structural changes in the close vicinity of dissipation in heat-sensitive structural elements, e.g. around the site where PBS is anchored to the membrane. This, in turn, brings about a diminishment in the energy supply from PBS to the photosystems and reorganization in the molecular architecture of PBS.