Zinc induced apoptotic death of mouse dendritic cells

Zinc ions (Zn²⁺) are food components with favourable effects in infectious disease. Zn²⁺ is taken up into dendritic cells (DCs), key players in the regulation of innate and adaptive immunity. In other cell types, Zn²⁺ has been shown to stimulate the formation of ceramide, which is in turn known to trigger suicidal cell death. The present study explored whether Zn²⁺ modifies ceramide formation and survival of bone marrow derived DCs. To this end, DCs were isolated from acid sphingomyelinase knockout (asm ^−/−) and corresponding wild type (asm ^+/+) mice and treated with different concentrations of Zn²⁺. Ceramide formation was assessed with anti-ceramide antibodies in FACS and immunohistochemical analysis, sub-G1 cell population by FACS analysis, break down of phosphatidylserine asymmetry by annexin V binding, cell death by propidium iodide incorporation, metabolic cell activity by MTT assay, ROS production from dichlorofluorescein fluorescence and activation of MAPKs by Western blotting. The treatment of asm ^+/+ DCs with low Zn²⁺ concentrations (up to 100 μM) was followed by ceramide formation, increase in sub-G1 cell population and phosphatidylserine exposure, effects blunted in asm ^−/− DCs. The treatment of DCs with C2-ceramide increased the percentage of sub-G1 and apoptotic DCs from both genotypes. Zn²⁺ led to similar activation of MAPKs in asm ^+/+ and asm ^−/− DCs and did not affect ROS production. Higher concentrations of Zn²⁺ led to a marked increase of propidium iodide incorporation in DCs of both genotypes. The present study reveals that in DCs Zn²⁺ triggers ceramide formation, which in turn compromises cell survival.     

RNase-sensitive and RNase-insensitive protective components isolated fromListeria monocytogenes

Crude ribosomes were isolated fromListeria monocytogenes serotype 4b and separated into two fractions by molecular sieve chromatography. Chemical analysis indicated that fraction I contained cell envelope components while fraction II contained the ribosomes. Both fractions protected mice againstListeria, but only in combination with the adjuvant dimethyldioctadecylammonium bromide (DDA). RNase-treatment; but not proteinase K-treatment destroyed the protective properties of fraction II, and RNA purified from fraction II also induced protection. Protection induced by fraction I was not affected by either RNase- or proteinase K-treatment. Both subcutaneous and intraperitoneal, but not intravenous administration of fraction I, fraction 11, or purified RNA induced significant protection against intraperitoneal infection, the intraperitoneal route of administration being the most effective. All preparations induced high levels of protection 3 to 7 days after administration, but protection was already decreased after 14 days. Protection induced with RNA appeared to be biphasic, because it also protected mice 1 day, but not 2 days after administration. Protection induced with both fraction I and RNA was at least in part nonspecific, because both preparations also protected mice againstL. monocytogenes serotype 3,Streptococcus pneumoniae andPseudomonas aeruginosa. Results are discussed in relation to previous work with analogous preparations fromP. aeruginosa.     

Dissecting out the Complex Ca2+-Mediated Phenylephrine-Induced Contractions of Mouse Aortic Segments

L-type Ca²⁺ channel (VGCC) mediated Ca²⁺ influx in vascular smooth muscle cells (VSMC) contributes to the functional properties of large arteries in arterial stiffening and central blood pressure regulation. How this influx relates to steady-state contractions elicited by α1-adrenoreceptor stimulation and how it is modulated by small variations in resting membrane potential (V_m) of VSMC is not clear yet. Here, we show that α1-adrenoreceptor stimulation of aortic segments of C57Bl6 mice with phenylephrine (PE) causes phasic and tonic contractions. By studying the relationship between Ca²⁺ mobilisation and isometric tension, it was found that the phasic contraction was due to intracellular Ca²⁺ release and the tonic contraction determined by Ca²⁺ influx. The latter component involves both Ca²⁺ influx via VGCC and via non-selective cation channels (NSCC). Influx via VGCC occurs only within the window voltage range of the channel. Modulation of this window Ca²⁺ influx by small variations of the VSMC V_m causes substantial effects on the contractile performance of aortic segments. The relative contribution of VGCC and NSCC to the contraction by α1-adrenoceptor stimulation could be manipulated by increasing intracellular Ca²⁺ release from non-contractile sarcoplasmic reticulum Ca²⁺ stores. Results of this study point to a complex interactions between α1-adrenoceptor-mediated VSMC contractile performance and Ca²⁺ release form contractile or non-contractile Ca²⁺ stores with concomitant Ca²⁺ influx. Given the importance of VGCC and their blockers in arterial stiffening and hypertension, they further point toward an additional role of NSCC (and NSCC blockers) herein.     

A Neutralization Epitope in the Hepatitis C Virus E2 Glycoprotein Interacts with Host Entry Factor CD81

The identification of a specific immunogenic candidate that will effectively activate the appropriate pathway for neutralizing antibody production is fundamental for vaccine design. By using a monoclonal antibody (1H8) that neutralizes HCV in vitro, we have demonstrated here that 1H8 recognized an epitope mapped between residues A524 and W529 of the E2 protein. We also found that the epitope residues A524, P525, Y527 and W529 were crucial for antibody binding, while the residues T526, Y527 and W529 within the same epitope engaged in the interaction with the host entry factor CD81. Furthermore, we detected “1H8-like” antibodies, defined as those with amino acid-specificity similar to 1H8, in the plasma of patients with chronic HCV infection. The time course study of plasma samples from Patient H, a well-characterized case of post-transfusion hepatitis C, showed that “1H8-like” antibodies could be detected in a sample collected almost two years after the initial infection, thus confirming the immunogenicity of this epitope in vivo. The characterization of this neutralization epitope with a function in host entry factor CD81 interaction should enhance our understanding of antibody-mediated neutralization of HCV infections.     

Progranulin antibodies entertain a proinflammatory environment in a subgroup of patients with psoriatic arthritis

Introduction

Psoriatic arthritis (PsA) is a distinctive inflammatory arthritis which may typically develop in a subgroup of individuals suffering from psoriasis. We recently described progranulin autoantibodies (PGRN-Abs) in the sera of patients with different autoimmune diseases including seronegative polyarthritis. In the present study we investigated the occurrence of PGRN-Abs in PsA.

Methods

PGRN-Abs were determined in 260 patients with PsA, 100 patients with psoriasis without arthritic manifestations (PsC) and 97 healthy controls using a recently described ELISA. PGRN plasma levels were determined from subgroups by a commercially available ELISA-kit. Possible functional effects of PGRN-antibodies were analysed in vitro by tumour necrosis factor (TNF)-α mediated cytotoxicity assays using WEHI-S and HT1080 cells.

Results

PGRN-Abs were detected with relevant titres in 50/260 (19.23%) patients with PsA, but in 0/100 patients with psoriasis without arthritic manifestations (P = 0.0001). All PGRN-Abs belonged to immunoglobulin G (IgG). PGRN-Abs were significantly more frequent in PsA patients with enthesitis or dactylitis. PGRN-Abs were also more frequent in PsA patients receiving treatment with TNF-α-blockers than in patients treated without TNF-α-blockers (20.8% versus 17.4%; P = 0.016). PGRN plasma levels were significantly lower in PGRN-Ab-positive patients with PsA than in healthy controls and patients with psoriasis without arthritic manifestations (P < 0.001), indicating a neutralizing effect of PGRN-Abs. Moreover cytotoxicity assays comparing PGRN-antibody positive with negative sera from matched patients with PsA, clearly showed a proinflammatory effect of PGRN antibodies.

Conclusion

Neutralizing PGRN-Abs occur with relevant titres in a subgroup of patients with PsA, but not in patients without arthritic manifestations (PsC). PGRN-Ab-positive patients had more frequent enthesitis or dactylitis. TNF-α-induced cytotoxicity assays demonstrated that the protective effects of progranulin were inhibited by serum containing PGRN-Abs. This suggests that PGRN-Ab might not only be useful as a diagnostic and prognostic marker, but may provide a proinflammatory environment in a subgroup of patients with PsA.     

Effects of introgression of two QTL for fusarium head blight resistance from Asian spring wheat by marker-assisted backcrossing into European winter wheat on fusarium head blight resistance,yield and quality traits

Breeding for fusarium head blight (FHB) resistance of wheat is a continuous challenge for plant breeders. Resistance to FHB is a quantitative trait, governed by several to many genes and modulated by environmental conditions. The presented study was undertaken to assess the effect on improving FHB resistance and on possible unwanted side effects (‘linkage drag’) of two resistance QTL, namely Fhb1 and Qfhs.ifa-5A, from the spring wheat line CM-82036 when transferred by marker-assisted backcrossing into several European winter wheat lines. To achieve these goals, we developed and evaluated fifteen backcross-two–derived families based on nine European winter wheat varieties as recipients and the FHB resistant variety CM-82036 as resistance donor. The QTL Qfhs.ifa-5A had a relatively small impact on increasing FHB resistance. On average lines with Fhb1 plus Qfhs.ifa-5A combined were only slightly more resistant compared to lines with Fhb1 alone. The obtained results suggest that the effect of the spring wheat–derived QTL on improving FHB resistance increases in the order Qfhs.ifa-5A < Fhb1 ≤ Qfhs.ifa-5A plus Fhb1 combined. The genetic background of the recipient line had a large impact on the resistance level of the obtained lines. No systematic negative effect of the spring wheat–derived QTL on grain yield, thousand grain weight, hectoliter weight and protein content was found. The use of spring wheat–derived FHB resistance QTL for breeding high yielding cultivars with improved FHB resistance appears therefore highly promising.     

Detection of a secreted metalloprotease within the nuclei of liver cells

ADAMTS13 is a secreted zinc metalloprotease expressed by various cell types. Here, we investigate its cellular pathway in endogenously expressing liver cell lines and after transient transfection with ADAMTS13. Besides compartmentalizations of the cellular secretory system, we detected an appreciable level of endogenous ADAMTS13 within the nucleus. A positively charged amino acid cluster (R-Q-R-Q-R-Q-R-R) present in the ADAMTS13 propeptide may act as a nuclear localization signal (NLS). Fusing this NLS-containing region to eGFP greatly potentiated its nuclear localization. Bioinformatics analysis suggests that the ADAMTS13 CUB-2 domain has a double-stranded beta helix (DSBH) structural architecture characteristic of various protein-protein interaction modules like nucleoplasmins, class I collagenase, tumor necrosis factor ligand superfamily, supernatant protein factor (SPF) and the B1 domain of neuropilin-2. Based on this contextual evidence and that largely conserved polar residues could be mapped on to a template CUB domain homolog, we hypothesize that a region in the ADAMTS13 CUB-2 domain with conserved polar residues might be involved in protein-protein interaction within the nucleus.     

Applying label-free dynamic mass redistribution technology to frame signaling of G protein-coupled receptors noninvasively in living cells

Label-free dynamic mass redistribution (DMR) is a cutting-edge assay technology that enables real-time detection of integrated cellular responses in living cells. It relies on detection of refractive index alterations on biosensor-coated microplates that originate from stimulus-induced changes in the total biomass proximal to the sensor surface. Here we describe a detailed protocol to apply DMR technology to frame functional behavior of G protein-coupled receptors that are traditionally examined with end point assays on the basis of detection of individual second messengers, such as cAMP, Ca(2+) or inositol phosphates. The method can be readily adapted across diverse cellular backgrounds (adherent or suspension), including primary human cells. Real-time recordings can be performed in 384-well microtiter plates and be completed in 2 h, or they can be extended to several hours depending on the biological question to be addressed. The entire procedure, including cell harvesting and DMR detection, takes 1-2 d.