Chloroplasts require glutathione reductase to balance reactive oxygen species and maintain efficient photosynthesis

Thiol‐based redox‐regulation is vital for coordinating chloroplast functions depending on illumination and has been throroughly investigated for thioredoxin‐dependent processes. In parallel, glutathione reductase (GR) maintains a highly reduced glutathione pool, enabling glutathione‐mediated redox buffering. Yet, how the redox cascades of the thioredoxin and glutathione redox machineries integrate metabolic regulation and detoxification of reactive oxygen species remains largely unresolved because null mutants of plastid/mitochondrial GR are embryo‐lethal in Arabidopsis thaliana. To investigate whether maintaining a highly reducing stromal glutathione redox potential (E_GSH) via GR is necessary for functional photosynthesis and plant growth, we created knockout lines of the homologous enzyme in the model moss Physcomitrella patens. In these viable mutant lines, we found decreasing photosynthetic performance and plant growth with increasing light intensities, whereas ascorbate and zeaxanthin/antheraxanthin levels were elevated. By in vivo monitoring stromal E_GSH dynamics, we show that stromal E_GSH is highly reducing in wild‐type and clearly responsive to light, whereas an absence of GR leads to a partial glutathione oxidation, which is not rescued by light. By metabolic labelling, we reveal changing protein abundances in the GR knockout plants, pinpointing the adjustment of chloroplast proteostasis and the induction of plastid protein repair and degradation machineries. Our results indicate that the plastid thioredoxin system is not a functional backup for the plastid glutathione redox systems, whereas GR plays a critical role in maintaining efficient photosynthesis.     

How Prof. Burnstock’s enthusiasm supported P2 receptor research in Germany

Purinergic Signalling -     

Bacterial Viability and Physical Properties of Antibacterially Modified Experimental Dental Resin Composites

Purpose

To investigate the antibacterial effect and the effect on the material properties of a novel delivery system with Irgasan as active agent and methacrylated polymerizable Irgasan when added to experimental dental resin composites.

Materials and Methods

A delivery system based on novel polymeric hollow beads, loaded with Irgasan and methacrylated polymerizable Irgasan as active agents were used to manufacture three commonly formulated experimental resin composites. The non-modified resin was used as standard (ST). Material A contained the delivery system providing 4 % (m/m) Irgasan, material B contained 4 % (m/m) methacrylated Irgasan and material C 8 % (m/m) methacrylated Irgasan. Flexural strength (FS), flexural modulus (FM), water sorption (WS), solubility (SL), surface roughness R_a, polymerization shrinkage, contact angle Θ, total surface free energy γ_S and its apolar γ_S ^LW, polar γ_S ^AB, Lewis acid γ_S ⁺and base γ_S ^- term as well as bacterial viability were determined. Significance was p < 0.05.

Results

The materials A to C were not unacceptably influenced by the modifications and achieved the minimum values for FS, WS and SL as requested by EN ISO 4049 and did not differ from ST what was also found for R_a. Only A had lower FM than ST. Θ of A and C was higher and γ_S ^AB of A and B was lower than of ST. Materials A to C had higher γ_S ⁺ than ST. The antibacterial effect of materials A to C was significantly increased when compared with ST meaning that significantly less vital cells were found.

Conclusion

Dental resin composites with small quantities of a novel antibacterially doped delivery system or with an antibacterial monomer provided acceptable physical properties and good antibacterial effectiveness. The sorption material being part of the delivery system can be used as a vehicle for any other active agent.     

Persistent Autoantibody-Production by Intermediates between Short-and Long-Lived Plasma Cells in Inflamed Lymph Nodes of Experimental Epidermolysis Bullosa Acquisita

Autoantibodies are believed to be maintained by either the continuous generation of short-lived plasma cells in secondary lymphoid tissues or by long-lived plasma cells localized in bone marrow and spleen. Here, we show in a mouse model for the autoimmune blistering skin disease epidermolysis bullosa acquisita (EBA) that chronic autoantibody production can also be maintained in inflamed lymph nodes, by plasma cells exhibiting intermediate lifetimes. After EBA induction by immunization with a mCOL7c-GST-fusion protein, antigen-specific plasma cells and CD4 T cells were analyzed. Plasma cells were maintained for months in stable numbers in the draining lymph nodes, but not in spleen and bone marrow. In contrast, localization of mCOL7c-GST -specific CD4 T cells was not restricted to lymph nodes, indicating that availability of T cell help does not limit plasma cell localization to this site. BrdU-incorporation studies indicated that pathogenic mCOL7c- and non-pathogenic GST-specific plasma cells resemble intermediates between short-and long-lived plasma cells with half-lives of about 7 weeks. Immunization with mCOL7c-GST also yielded considerable numbers of plasma cells neither specific for mCOL7c- nor GST. These bystander-activated plasma cells exhibited much shorter half-lives and higher population turnover, suggesting that plasma cell lifetimes were only partly determined by the lymph node environment but also by the mode of activation. These results indicate that inflamed lymph nodes can harbor pathogenic plasma cells exhibiting distinct properties and hence may resemble a so far neglected site for chronic autoantibody production.     

TGFβs Modulate Permeability of the Blood-Epididymis Barrier in an In Vitro Model

The blood-epididymis barrier (BEB) is formed by epithelial tight junctions mediating selective permeability of the epididymal epithelium. Defective barrier function can disturb the balance of the epididymal milieu, which may result in infertility. The stroma of the epididymis contains high amounts of cytokines of the TGFβ family of unknown function. We screened possible effects of all three TGFβ isoforms on paracellular tightness in a BEB in vitro model based on the strongly polarized mouse epididymal epithelial MEPC5 cells in the transwell system. In this model we found a robust transepithelial electrical resistance (TER) of about 840 Ω x cm². Effects on the paracellular permeability were evaluated by two methods, TER and FITC-Dextran-based tracer diffusion assays. Both assays add up to corresponding results indicating a time-dependent disturbance of the BEB differentially for the three TGFβ isoforms (TGFβ3>TGFβ1>TGFβ2) in a TGFβ-recetor-1 kinase- and Smad-dependent manner. The tight junction protein claudin-1 was found to be reduced by the treatment with TGFβs, whereas occludin was not influenced. Epididymal epithelial cells are predominantly responsive to TGFβs from the basolateral side, suggesting that TGFβ may have an impact on the epididymal epithelium from the stroma in vivo. Our data show for the first time that TGFβs decrease paracellular tightness in epididymal epithelial cells, thus establishing a novel mechanism of regulation of BEB permeability, which is elementary for sperm maturation and male fertility.     

An automated immunoassay for early specificity profiling of antibodies

Antibody-based therapeutics are of great value for the treatment of human diseases. In addition to functional activity, affinity or physico-chemical properties, antibody specificity is considered to be one of the most crucial attributes for safety and efficacy. Consequently, appropriate studies are required before entering clinical trials.

High content protein arrays are widely applied to assess antibody specificity, but this commercial solution can only be applied to final therapeutic antibody candidates because such arrays are expensive and their throughput is limited. A flexible, high-throughput and economical assay that allows specificity testing of IgG or Fab molecules during early discovery is described here. The 384-well microtiter plate assay contains a comprehensive panel of 32 test proteins and uses electrochemiluminescence as readout.

The Protein Panel Profiling (3P) was used to analyze marketed therapeutic antibodies that all showed highly specific binding profiles. Subsequently, 3P was applied to antibody candidates from early discovery and the results compared well with those obtained with a commercially available high content protein chip. Our results suggest that 3P can be applied as an additional filter for lead selection, allowing the identification of favorable antibody candidates in early discovery and thereby increasing the speed and possibility of success in drug development.     

Growth of Arabidopsis seedlings on high fungal doses of Piriformospora indica has little effect on plant performance,stress, and defense gene expression in spite of elevated jasmonic acid and jasmonic acid-isoleucine levels in the roots

The endophytic fungus Piriformospora indica colonizes the roots of many plant species including Arabidopsis and promotes their performance, biomass, and seed production as well as resistance against biotic and abiotic stress. Imbalances in the symbiotic interaction such as uncontrolled fungal growth result in the loss of benefits for the plants and activation of defense responses against the microbe. We exposed Arabidopsis seedlings to a dense hyphal lawn of P. indica. The seedlings continue to grow, accumulate normal amounts of chlorophyll, and the photosynthetic parameters demonstrate that they perform well. In spite of high fungal doses around the roots, the fungal material inside the roots was not significantly higher when compared with roots that live in a beneficial symbiosis with P. indica. Fifteen defense- and stress-related genes including PR2, PR3, PAL2, and ERF1 are only moderately upregulated in the roots on the fungal lawn, and the seedlings did not accumulate H₂O₂/radical oxygen species. However, accumulation of anthocyanin in P. indica-exposed seedlings indicates stress symptoms. Furthermore, the jasmonic acid (JA) and jasmonic acid-isoleucine (JA-Ile) levels were increased in the roots, and consequently PDF1.2 and a newly characterized gene for a 2-oxoglurate and Fe²⁺-dependent oxygenase were upregulated more than 7-fold on the dense fungal lawn, in a JAR1- and EIN3-dependent manner. We conclude that growth of A. thaliana seedlings on high fungal doses of P. indica has little effect on the overall performance of the plants although elevated JA and JA-Ile levels in the roots induce a mild stress or defense response.     

Replication Vesicles are Load- and Choke-Points in the Hepatitis C Virus Lifecycle

Hepatitis C virus (HCV) infection develops into chronicity in 80% of all patients, characterized by persistent low-level replication. To understand how the virus establishes its tightly controlled intracellular RNA replication cycle, we developed the first detailed mathematical model of the initial dynamic phase of the intracellular HCV RNA replication. We therefore quantitatively measured viral RNA and protein translation upon synchronous delivery of viral genomes to host cells, and thoroughly validated the model using additional, independent experiments. Model analysis was used to predict the efficacy of different classes of inhibitors and identified sensitive substeps of replication that could be targeted by current and future therapeutics. A protective replication compartment proved to be essential for sustained RNA replication, balancing translation versus replication and thus effectively limiting RNA amplification. The model predicts that host factors involved in the formation of this compartment determine cellular permissiveness to HCV replication. In gene expression profiling, we identified several key processes potentially determining cellular HCV replication efficiency.