The Ca2+-binding protein calretinin is selectively enriched in a subpopulation of the epithelial rests of Malassez

During tooth development, the inner and outer enamel epithelia fuse by mitotic activity to produce a bilayered epithelial sheath termed Hertwig’s epithelial root sheath (HERS). The epithelial rests of Malassez (ERM) are the developmental residues of HERS and remain in the adult periodontal ligament (PDL). Although the cellular regulation of the Ca²⁺-binding proteins parvalbumin, calbindin-D28k, and calretinin has been reported in the inner and outer enamel epithelia during tooth development, an involvement of Ca²⁺-binding proteins in the ERM has not so far been characterized. Among the three Ca²⁺-binding proteins tested (calbindin D28k, parvalbumin, calretinin), we have only been able to detect calretinin in a subpopulation of adult rat molar ERM, by using quantitative immunohistochemical and confocal immunofluorescence techniques. TrkA (a marker for ERM) is present in numerous epithelial cell clusters, whereas calretinin has been localized in the cytosol and perinuclear region of a subpopulation of TrkA-positive cells. We conclude that, in inner and outer enamel epithelial cells, Ca²⁺ is regulated by calbindin, parvalbumin, and calretinin during tooth development, whereas in the ERM of adult PDL, Ca²⁺ is regulated only by calretinin. The expression of Ca²⁺-binding proteins is restricted in a developmental manner in the ERM.     

Interplay between influenza A virus and the innate immune signaling

Pathogens such as influenza A viruses (IAV) have to overcome a number of barriers defined and maintained by the host, to successfully establish an infection. One of the initial barriers is collectively characterized as the innate immune system. This is a broad anti-pathogen defense program that ranges from the action of natural killer cells to the induction of an antiviral cytokine response. In this article we will focus on new developments and discoveries concerning the interaction of IAV with the cellular innate immune signaling. We discuss new mechanisms of interference of IAV with the pathogen recognition receptor RIG-I and the type I IFN antagonist NS1 in the background of already known and established concepts. Further we summarize progress related to recently identified IFN induced proteins and the role of RNA interference in the context of IAV infection.     

Improved Strains and Plasmid Vectors for Conditional Overexpression of His-Tagged Proteins in Haloferax volcanii

Research into archaea will not achieve its full potential until systems are in place to carry out genetics and biochemistry in the same species. Haloferax volcanii is widely regarded as the best-equipped organism for archaeal genetics, but the development of tools for the expression and purification of H. volcanii proteins has been neglected. We have developed a series of plasmid vectors and host strains for conditional overexpression of halophilic proteins in H. volcanii. The plasmids feature the tryptophan-inducible p.tnaA promoter and a 6×His tag for protein purification by metal affinity chromatography. Purification is facilitated by host strains, where pitA is replaced by the ortholog from Natronomonas pharaonis. The latter lacks the histidine-rich linker region found in H. volcanii PitA and does not copurify with His-tagged recombinant proteins. We also deleted the mrr restriction endonuclease gene, thereby allowing direct transformation without the need to passage DNA through an Escherichia coli dam mutant.Over the past century, our understanding of fundamental biological processes has grown exponentially, and this would have been impossible without the use of organisms that are amenable to experimental manipulation. Model species, such as Escherichia coli, Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, Mus musculus, and Arabidopsis thaliana, have become a byword for scientific progress (15). The rational choice of a model organism is critically important, and certain features are taken for granted, such as ease of cultivation, a short generation time, and systems for genetic manipulation. This list has now grown to include a genome sequence and methods for biochemical analysis of purified proteins in vitro.Research into archaea has lagged behind work on bacteria and eukaryotes but has nonetheless yielded profound insights (2). One hurdle has been the paucity of archaeal organisms suitable for both biochemistry and genetics. For example, Methanothermobacter thermautotrophicus is a stalwart of archaeal biochemistry but has proved resistant to even the most rudimentary genetic manipulation (2). Progress has recently been made with another biochemical workhorse, Sulfolobus spp., and a few genetic tools are now available (6, 13, 37). Methanosarcina spp. and Thermococcus kodakaraensis offer alternative systems with an increasing array of techniques (16, 35, 36), but sophisticated genetics has traditionally been the preserve of haloarchaea, of which Haloferax volcanii is the organism of choice (39). It is easy to culture, the genome has been sequenced (19), and there are several selectable markers and plasmids for transformation and gene knockout (3, 7, 31), including a Gateway system (14), as well as reporter genes (20, 33) and a tightly controlled inducible promoter (26).The genetic prowess of H. volcanii is not yet fully matched by corresponding systems for protein overexpression and purification. Like other haloarchaea, H. volcanii grows in high salt concentrations (2 to 5 M NaCl), and to cope with the osmotic potential of such environments, it accumulates high intracellular concentrations of potassium ions (12). Consequently, halophilic proteins are adapted to function at high salt concentrations and commonly feature a large excess of acidic amino acids; the negative surface charge is thought to be critical to solubility (28). This can pose problems for expression in heterologous hosts, such as E. coli, since halophilic proteins can misfold and aggregate under conditions of low ionic strength. The purification of misfolded halophilic enzymes from E. coli has relied on the recovery of insoluble protein from inclusion bodies, followed by denaturation and refolding in hypersaline solutions (8, 11). This approach is feasible only where the protein is well characterized and reconstitution of the active form can be monitored (for example, by an enzymatic assay). Furthermore, archaeal proteins expressed in heterologous bacterial hosts lack posttranslational modifications, such as acetylation or ubiquitination (4, 22), which are critical to understanding their biological function.Systems for expression of halophilic proteins in a native haloarchaeal host are therefore required. A number of studies have successfully purified recombinant proteins with a variety of affinity tags after overexpression in H. volcanii. For example, Humbard et al. employed tandem affinity tagging to purify 20S proteasomal core particles from the native host (23). However, the protein expression constructs used in these studies were custom made and somewhat tailored to the application in question. We report here the development of “generic” plasmid vectors and host strains for conditional overexpression of halophilic proteins in H. volcanii. The plasmids feature a tryptophan-inducible promoter derived from the tnaA gene of H. volcanii (26). We demonstrate the utility of these vectors by overexpressing a hexahistidine-tagged recombinant version of the H. volcanii RadA protein. Purification was greatly facilitated by a host strain in which the endogenous pitA gene was replaced by an ortholog from Natronomonas pharaonis. The latter protein lacks the histidine-rich linker region found in H. volcanii PitA (5) and therefore does not copurify with His-tagged recombinant proteins. Finally, we deleted the mrr gene of H. volcanii, which encodes a restriction enzyme that cleaves foreign DNA methylated at GATC residues. The mrr deletion strain allows direct transformation of H. volcanii without the need to passage plasmid DNA through an E. coli dam mutant (21).     

Spin labeling of the Escherichia coli NADH ubiquinone oxidoreductase (complex I)

The proton-pumping NADH:ubiquinone oxidoreductase, the respiratory complex I, couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. Electron microscopy revealed the two-part structure of the complex with a peripheral arm involved in electron transfer and a membrane arm most likely involved in proton translocation. It was proposed that the quinone binding site is located at the joint of the two arms. Most likely, proton translocation in the membrane arm is enabled by the energy of the electron transfer reaction in the peripheral arm transmitted by conformational changes. For the detection of the conformational changes and the localization of the quinone binding site, we set up a combination of site-directed spin labeling and EPR spectroscopy. Cysteine residues were introduced to the surface of the Escherichia coli complex I. The spin label (1-oxyl-2,2,5,5-tetramethyl-Δ3-pyrroline-3-methyl)-methanethiosulfonate (MTSL) was exclusively bound to the engineered positions. Neither the mutation nor the labeling had an effect on the NADH:decyl-ubiquinone oxidoreductase activity. The characteristic signals of the spin label were detected by EPR spectroscopy, which did not change by reducing the preparation with NADH. A decyl-ubiquinone derivative with the spin label covalently attached to the alkyl chain was synthesized in order to localize the quinone binding site. The distance between a MTSL labeled complex I variant and the bound quinone was determined by continuous-wave (cw) EPR allowing an inference on the location of the quinone binding site. The distances between the labeled quinone and other complex I variants will be determined in future experiments to receive further geometry information by triangulation.     

Increased expression of costimulatory markers CD134 and CD80 on interleukin-17 producing T cells in patients with systemic lupus erythematosus

Introduction  

There is growing evidence that interleukin 17 (IL-17) producing T cells are involved in the pathogenesis of systemic lupus erythematosus (SLE). Previous studies showed that increased percentages of T-cell subsets expressing the costimulatory molecules CD80 and CD134 are associated with disease activity and renal involvement in SLE. The aim of this study was to investigate the distribution and phenotypical characteristics of IL-17 producing T-cells in SLE, in particular in patients with lupus nephritis, with emphasis on the expression of CD80 and CD134.