Oscillations in photosynthesis are initiated and supported by imbalances in the supply of ATP and NADPH to the Calvin cycle

Oscillations in the rate of photosynthesis of sunflower (Helianthus annuus L.) leaves were induced by subjecting leaves, whose photosynthetic apparatus had been activated, to a sudden transition from darkness or low light to high-intensity illumination, or by transfering them in the light from air to an atmosphere containing saturating CO₂. It was found that at the first maximum, light-and CO₂-saturated photosynthesis can be much faster than steady-state photosynthesis. Both Q_A in the reaction center of PS II and P₇₀₀ in the reaction center of PS I of the chloroplast electron-transport chain were more oxidized during the maxima of photosynthesis than during the minima. Maxima of P₇₀₀ oxidation slightly preceded maxima in photosynthesis. During a transition from low to high irradiance, the assimilatory force F_A, which was calculated from ratios of dihydroxyacetone phosphate to phosphoglycerate under the assumption that the reactions catalyzed by NADP-dependent glyceraldehydephosphate dehydrogenase, phosphoglycerate kinase and triosephosphate isomerase are close to equilibrium, oscillated in parallel with photosynthesis. However, only one of its components, the calculated phosphorylation potential (ATP)/(ADP)(P_i), paralleled photosynthesis, whereas calculated NADPH/NADP ratios exhibited antiparallel behaviour. When photosynthetic oscillations were initiated by a transition from low to high CO₂, the assimilatory force F_A declined, was very low at the first minimum of photosynthesis and increased as photosynthesis rose to its second maximum. The observations indicate that the minima in photosynthesis are caused by lack of ATP. This leads to overreduction of the electron-transport chain which is indicated by the reduction of P₇₀₀. During photosynthetic oscillations the chloroplast thylakoid system is unable to adjust the supply of ATP and NADPH rapidly to demand at the stoichiometric relationship required by the carbonreduction cycle.Abbreviations PGA 3-phosphoglycerate - DHAP dihydroxyacetone phosphate - P₇₀₀ electron-donor pigment in the reaction enter of PS I - Q_A quinone acceptor in the reaction center of PS II This work received support from the Estonian Academy of Sciences, the Bavarian Ministry of Science and Art and the Sonderforschungsbereich 251 of the University of Würzburg. We are grateful for criticism by D.A. Walker, Robert Hill Institute, University of Sheffield, U.K. and by Mark Stitt, Institute of Botany, University of Heidelberg, FRG.     

Mucin biopolymers as broad-spectrum antiviral agents

Mucus is a porous biopolymer matrix that coats all wet epithelia in the human body and serves as the first line of defense against many pathogenic bacteria and viruses. However, under certain conditions viruses are able to penetrate this infection barrier, which compromises the protective function of native mucus. Here, we find that isolated porcine gastric mucin polymers, key structural components of native mucus, can protect an underlying cell layer from infection by small viruses such as human papillomavirus (HPV), Merkel cell polyomavirus (MCV), or a strain of influenza A virus. Single particle analysis of virus mobility inside the mucin barrier reveals that this shielding effect is in part based on a retardation of virus diffusion inside the biopolymer matrix. Our findings suggest that purified mucins may be used as a broad-range antiviral supplement to personal hygiene products, baby formula or lubricants to support our immune system.     

Altering an artificial Gagpolnef polyprotein and mode of ENV co-administration affects the immunogenicity of a clade C HIV DNA vaccine

HIV-1 candidate vaccines expressing an artificial polyprotein comprising Gag, Pol and Nef (GPN) and a secreted envelope protein (Env) were shown in recent Phase I/II clinical trials to induce high levels of polyfunctional T cell responses; however, Env-specific responses clearly exceeded those against Gag. Here, we assess the impact of the GPN immunogen design and variations in the formulation and vaccination regimen of a combined GPN/Env DNA vaccine on the T cell responses against the various HIV proteins. Subtle modifications were introduced into the GPN gene to increase Gag expression, modify the expression ratio of Gag to PolNef and support budding of virus-like particles. I.m. administration of the various DNA constructs into BALB/c mice resulted in an up to 10-fold increase in Gag- and Pol-specific IFNγ(+) CD8(+) T cells compared to GPN. Co-administering Env with Gag or GPN derivatives largely abrogated Gag-specific responses. Alterations in the molar ratio of the DNA vaccines and spatially or temporally separated administration induced more balanced T cell responses. Whereas forced co-expression of Gag and Env from one plasmid induced predominantly Env-specific T cells responses, deletion of the only H-2(d) T cell epitope in Env allowed increased levels of Gag-specific T cells, suggesting competition at an epitope level. Our data demonstrate that the biochemical properties of an artificial polyprotein clearly influence the levels of antigen-specific T cells, and variations in formulation and schedule can overcome competition for the induction of these responses. These results are guiding the design of ongoing pre-clinical and clinical trials.     

Identification of specific miRNAs targeting proteins of the apical junctional complex that simulate the probiotic effect of E. coli Nissle 1917 on T84 epithelial cells

In the intestine, dysregulation of miRNA is associated with inflammation, disruption of the gastrointestinal barrier, and the onset of gastrointestinal disorders. This study identifies miRNAs involved in the maintenance of intercellular junctions and barrier integrity. For the functional identification of barrier affecting miRNAs, we took advantage of the barrier-enforcing effects of the probiotic bacterium Escherichia coli Nissle 1917 (EcN) which can be monitored by enhanced transepithelial resistance (TER). miRNA-profiling of T84 monolayers prior and after co-incubation with EcN revealed for the first time differentially regulated miRNAs (miR-203, miR-483-3p, miR-595) targeting tight junction (TJ) proteins. Using real-time PCR, Western blotting and specific miRNA mimics, we showed that these miRNAs are involved in the regulation of barrier function by modulating the expression of regulatory and structural components of tight junctional complexes. Furthermore, specific inhibitors directed at these miRNA abrogated the disturbance of tight junctions induced by enteropathogenic E. coli (EPEC). The half-maximal inhibitory concentration (IC(50)) was determined to 340 nM by monitoring inhibitor kinetics. In summary, we conclude that specific miRNAs effect regulatory as well as structural proteins of the junctional complex which in turn are involved in the barrier enhancing effect of EcN. Hence, we suggest that the application of miRNAs might be refined and further developed as a novel supportive strategy for the treatment of gastrointestinal disorders.     

Effect of salinity changes on the bacterial diversity, photosynthesis and oxygen consumption of cyanobacterial mats from an intertidal flat of the Arabian Gulf

The effects of salinity fluctuation on bacterial diversity, rates of gross photosynthesis (GP) and oxygen consumption in the light (OCL) and in the dark (OCD) were investigated in three submerged cyanobacterial mats from a transect on an intertidal flat. The transect ran 1 km inland from the low water mark along an increasingly extreme habitat with respect to salinity. The response of GP, OCL and OCD in each sample to various salinities (65 per thousand, 100 per thousand, 150 per thousand and 200 per thousand) were compared. The obtained sequences and the number of unique operational taxonomic units showed clear differences in the mats' bacterial composition. While cyanobacteria decreased from the lower to the upper tidal mat, other bacterial groups such as Chloroflexus and Cytophaga/Flavobacteria/Bacteriodetes showed an opposite pattern with the highest dominance in the middle and upper tidal mats respectively. Gross photosynthesis and OCL at the ambient salinities of the mats decreased from the lower to the upper tidal zone. All mats, regardless of their tidal location, exhibited a decrease in areal GP, OCL and OCD rates at salinities > 100 per thousand. The extent of inhibition of these processes at higher salinities suggests an increase in salt adaptation of the mats microorganisms with distance from the low water line. We conclude that the resilience of microbial mats towards different salinity regimes on intertidal flats is accompanied by adjustment of the diversity and function of their microbial communities.     

Definition of immunogenic carbohydrate epitopes

Carbohydrates are known as sources of immunological cross-reactivity of allergenic significance. In celery and in cypress pollen, the major allergens Api g 5 and Cup a 1 are recognised by antisera raised against anti-horseradish peroxidase and by patients' IgE which apparently bind carbohydrate epitopes; mass spectrometric analysis of the tryptic peptides and of their N-glycans showed the presence of oligosaccharides carrying both xylose and core alpha1,3-fucose residues. Core alpha1,3-fucose residues are also a feature of invertebrates: genetic and biochemical studies on the fruitfly Drosophila melanogaster, the parasitic trematode Schistosoma mansoni and the nematode worm Caenorhabditis elegans indicate that these organisms possess core alpha1,3-fucosyltransferases. Various experiments have shown that fucosyltransferases from both fly and worm are responsible in vivo and in vitro for the synthesis of N-glycans which cross-react with anti-horseradish peroxidase; thus, we can consider these enzymes as useful tools in generating standard compounds for testing cross-reactive carbohydrate epitopes of allergenic interest.     

Responses of Antarctic Iridaea cordata (Rhodophyta) tetraspores exposed to ultraviolet radiation

In Antarctica ozone depletion is highest during spring, coinciding with the reproduction of many seaweed species. Propagules are the life-stage of an alga most susceptible to environmental perturbations. Therefore, fertile thalli of Iridaea cordata (Turner) Bory (Rhodophyta) were collected in the eulittoral of King George Island (Antarctica) to examine spore susceptibility to ultraviolet radiation (UVR). In the laboratory, freshly released tetraspores were exposed to photosynthetically active radiation (PAR) (400–700 nm), PAR+UV-A (320–700 nm) or PAR+UV-A+UV-B (280–700 nm). Photosynthetic efficiency was measured during 1–8 h of exposure and after 48 h of recovery. Additionally, mycosporine-like amino acids (MAAs) and DNA damage were determined. Saturating irradiance of photosynthesis of freshly released tetraspores was 57 µmol photons m⁻² s⁻¹. Exposure to increasing fluence of PAR reduced photosynthetic efficiency. UVR further decreased the photosynthetic efficiencies of the tetraspores but spores were able to recover completely after UVR exposure and 2 days post-cultivation under low PAR. DNA damage was minimal and lesions were effectively repaired under photoreactivating light. Concentrations of the MAAs shinorine and palythine were higher in tetraspores treated with UVR than in spores only exposed to PAR. Generally, the tetraspores show a good UV tolerance. This flexible response of the tetraspores of this species to changing radiation conditions enables the alga to grow along a considerable depth gradient from the sublittoral to the eulittoral where they can be exposed to enhanced UVBR under conditions of stratospheric ozone depletion.     

Fibrin Clot Structure and Platelet Aggregation in Patients with Aspirin Treatment Failure

Background

Aspirin is a cornerstone in prevention of cardiovascular events and modulates both platelet aggregation and fibrin clot formation. Some patients experience cardiovascular events whilst on aspirin, often termed aspirin treatment failure (ATF). This study evaluated both platelet aggregation and fibrin clot structure in patients with ATF.

Methods

We included 177 stable coronary artery disease patients on aspirin monotherapy. Among these, 116 (66%) had ATF defined as myocardial infarction (MI) whilst on aspirin. Platelet aggregation was assessed by Multiplate® aggregometry and VerifyNow®, whereas turbidimetric assays and scanning electron microscopy were employed to study fibrin clot characteristics.

Results

Enhanced platelet aggregation was observed in patients with ATF compared with non-MI patients following stimulation with arachidonic acid 1.0 mM (median 161 (IQR 95; 222) vs. 97 (60; 1776) AU*min, p = 0.005) and collagen 1.0 µg/mL (293 (198; 427) vs. 220 (165; 370) AU*min, p = 0.03). Similarly, clot maximum absorbance, a measure of fibrin network density, was increased in patients with ATF (0.48 (0.41; 0.52) vs. 0.42 (0.38; 0.50), p = 0.02), and this was associated with thinner fibres (mean ± SD: 119.7±27.5 vs. 127.8±31.1 nm, p = 0.003) and prolonged lysis time (552 (498; 756) vs. 519 (468; 633) seconds; p = 0.02). Patients with ATF also had increased levels of C-reactive protein (CRP) (1.34 (0.48; 2.94) and 0.88 (0.32; 1.77) mg/L, p = 0.01) compared with the non-MI group. Clot maximum absorbance correlated with platelet aggregation (r = 0.31–0.35, p-values<0.001) and CRP levels (r = 0.60, p<0.001).

Conclusions

Patients with aspirin treatment failure showed increased platelet aggregation and altered clot structure with impaired fibrinolysis compared with stable CAD patients without previous MI. These findings suggest that an increased risk of aspirin treatment failure may be identified by measuring both platelet function and fibrin clot structure.     

3-Hydroxypropionaldehyde production from crude glycerol by <Emphasis Type="Italic">Lactobacillus diolivorans</Emphasis> with enhanced glycerol uptake

Background

In their quest for sustainable development and effective management of greenhouse gas emissions, our societies pursue a shift away from fossil-based resources towards renewable resources. With 95% of our current transportation energy being petroleum based, the application of alternative, carbon-neutral products—among them biodiesel—is inevitable. In order to enhance the cost structure of biodiesel biorefineries, the valorization of the crude glycerol waste stream into high-value platform chemicals is of major importance.

Results

The purpose of this study is the production of 3-hydroxypropionaldehyde (3-HPA) from biodiesel-derived crude glycerol by Lactobacillus diolivorans. Particular focus is given on overcoming potential limitations of glycerol transport into the cell, in order to use the cells’ total glycerol dehydratase capability towards the formation of 3-HPA as the main product. Recombinant overexpression of the endogenous glycerol uptake facilitating protein PduF results in a significant increase of glycerol conversion by a factor of 1.3. Concomitantly, glycerol dehydratase activity increased from initially 1.70 ± 0.03 U/mg protein to 2.23 ± 0.11 U/mg protein. With this approach, an average productivity of 4.8 g_3-HPA/(g_CDM h) yielding up to 35.9 g/L 3-HPA and 0.91 mol_3-HPA/mol_Glycerol have been obtained.

Conclusion

Lactobacillus diolivorans proves to be a valuable cell factory for the utilization of crude glycerol delivering high-value C3 chemicals like 3-HPA, 1,3-propanediol (1,3-PDO) and 3-hydroxypropionic acid (3-HP). Enhancing the glycerol influx into the cell by genetic engineering was successful paving the way towards the commercial production of 3-HPA.

     

The cell surface protein MUL_3720 confers binding of the skin pathogen Mycobacterium ulcerans to sulfated glycans and keratin

Mycobacterium ulcerans is the causative agent of the chronic, necrotizing skin disease Buruli ulcer. Modes of transmission and molecular mechanisms involved in the establishment of M. ulcerans infections are poorly understood. Interactions with host glycans are often crucial in bacterial pathogenesis and the 22 kDa M. ulcerans protein MUL_3720 has a putative role in host cell attachment. It has a predicted N-terminal lectin domain and a C-terminal peptidoglycan-binding domain and is highly expressed on the surface of the bacilli. Here we report the glycan-binding repertoire of whole, fixed M. ulcerans bacteria and of purified, recombinant MUL_3720. On an array comprising 368 diverse biologically relevant glycan structures, M. ulcerans cells showed binding to 64 glycan structures, representing several distinct classes of glycans, including sulfated structures. MUL_3720 bound only to glycans containing sulfated galactose and GalNAc, such as glycans known to be associated with keratins isolated from human skin. Surface plasmon resonance studies demonstrated that both whole, fixed M. ulcerans cells and MUL_3720 show high affinity interactions with both glycans and human skin keratin extracts. This MUL_3720-mediated interaction with glycans associated with human skin keratin may contribute to the pathobiology of Buruli ulcer.