Nonstructural proteins NS2-3 and NS4A of classical swine fever virus: essential features for infectious particle formation

The nonstructural protein NS2-3 of pestiviruses undergoes tightly regulated processing. For bovine viral diarrhea virus it was shown that uncleaved NS2-3 is required for infectious particle formation while cleaved NS3 is essential for genome replication. To further investigate the functions of NS2-3 and NS4A in the pestivirus life cycle, we established T7 RNA polymerase-dependent trans-complementation for p7-NS2-3-4A of classical swine fever virus (CSFV). Expression of NS2-3 and NS4A in trans restored the production of infectious particles from genomes lacking NS2-3 expression. Co-expression of cleaved NS4A was essential. None of the enzymatic activities harbored by NS2-3 were required for infectious particle formation. Importantly, expression of uncleavable NS2-3 together with NS4A rescued infectious particles from a genome lacking NS2, demonstrating that cleaved NS2 per se has no additional essential function. These data indicate that NS2-3 and NS3, each in association with NS4A, have independent functions in the CSFV life cycle.

Immunodetection of 3-nitrotyrosine in the liver of zymosan-treated rats with a new monoclonal antibody: comparison to analysis by HPLC

Zymosan-induced peritonitis is associated with an increased production of reactive nitrogen oxides that may contribute to the often-observed failure of multiple organ systems in this model of acute inflammation. Quantitative biochemical evidence is provided for a marked 13-fold increase in protein-bound 3-nitrotyrosine (NTyr), a biomarker of reactive nitrogen oxides, in liver tissue of zymosan-treated rats. In order to investigate the localization of NTyr in this affected tissue, a monoclonal antibody, designated 39B6, was raised against 3-(4-hydroxy-3-nitrophenylacetamido) propionic acid-bovine serum albumin conjugate and its performance characterized. 39B6 was judged by competition ELISA to be approximately 2 orders of magnitude more sensitive than a commercial anti-NTyr monoclonal antibody. Binding characteristics of 39B6 were similar, but not identical, to that of a commercial affinity-purified polyclonal antibody in ELISA and immunohistochemical analyses. Western blot experiments revealed high specificity of 39B6 against NTyr and increased immunoreactivity of specific proteins from liver tissue homogenates of zymosan-treated rats. Immunohistochemical analysis of liver sections indicated a marked zymosan-induced increase in immunofluorescent staining, which was particularly intense in or adjacent to nonparenchymal cells, but not in the parenchymal cells of this tissue. Quantitative analysis of fractions enriched in these cell populations corroborated the immunofluorescent data, although the relative amounts detected in response to zymosan treatment was greatly reduced compared to whole liver tissue. These results demonstrate the high specificity of the newly developed antibody and its usefulness in Western blot and immunohistochemical analysis for NTyr, confirm the presence of NTyr by complementary methods, and suggest the possible involvement of reactive nitrogen oxides in hepatic vascular dysfunction.

Claudin-1 induced sealing of blood-brain barrier tight junctions ameliorates chronic experimental autoimmune encephalomyelitis

In experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS), loss of the blood-brain barrier (BBB) tight junction (TJ) protein claudin-3 correlates with immune cell infiltration into the CNS and BBB leakiness. Here we show that sealing BBB TJs by ectopic tetracycline-regulated expression of the TJ protein claudin-1 in Tie-2 tTA//TRE-claudin-1 double transgenic C57BL/6 mice had no influence on immune cell trafficking across the BBB during EAE and furthermore did not influence the onset and severity of the first clinical disease episode. However, expression of claudin-1 did significantly reduce BBB leakiness for both blood borne tracers and endogenous plasma proteins specifically around vessels expressing claudin-1. In addition, mice expressing claudin-1 exhibited a reduced disease burden during the chronic phase of EAE as compared to control littermates. Our study identifies BBB TJs as the critical structure regulating BBB permeability but not immune cell trafficking into CNS during EAE, and indicates BBB dysfunction is a potential key event contributing to disease burden in the chronic phase of EAE. Our observations suggest that stabilizing BBB barrier function by therapeutic targeting of TJs may be beneficial in treating MS, especially when anti-inflammatory treatments have failed.

High expression of FOXP3 in primary melanoma is associated with tumour progression.

BACKGROUND The antitumour immune response plays an important role in the prognosis of melanoma. High numbers of circulating regulatory T cells have been associated with rapid disease progression. OBJECTIVES To assess the influence of forkhead box protein (FOXP)3, CD1a and langerin expression on the prognosis of primary melanoma. METHODS We analysed 185 primary melanomas by immunohistochemical staining for expression of the regulatory T-cell marker FOXP3 and the dendritic cell markers langerin and CD1a, and correlated marker expression with clinical outcome. RESULTS Disease-free survival and overall survival were significantly longer in patients expressing low levels of FOXP3 in the primary melanoma, whereas they were associated with high expression of CD1a. The negative prognostic value of FOXP3 expression was independent of the Breslow tumour thickness. Langerin expression did not correlate with the clinical outcome. CONCLUSIONS High expression of FOXP3 in the primary melanoma may be used as an additional independent prognostic marker for early tumour progression in patients with melanoma.

The ribosome as novel target for stress-induced small regulatory RNAs

Small non-protein-coding RNA (ncRNA) molecules represent major contributors to regulatory networks in controlling gene expression in a highly efficient manner. All of the recently discovered regulatory ncRNAs that act on translation (e.g. microRNAs, siRNAs or antisense RNAs) target the mRNA rather than the ribosome. To address the question, whether small ncRNA regulators exist that are capable of modulating the rate of protein production by directly interacting with the ribosome, we have analyzed the small ncRNA interactomes of ribosomes Deep-sequencing and subsequent bioinformatic analyses revealed thousands of putative ribosome-associated ncRNAs in various model organisms (1,2). For a subset of these ncRNA candidates we have gathered experimental evidence that they associate with ribosomes in a stress-dependent manner and are capable of regulating gene expression by fine-tuning the rate of protein biosynthesis (3,4). Many of the investigated ribosome-bound small ncRNA appear to be processing products from larger functional RNAs, such as tRNAs (2,3) or mRNAs (3). Post-transcriptional cleavage of RNA molecules to generate smaller fragments is a widespread mechanism that enlarges the structural and functional complexity of cellular RNomes. Our data reveal the ribosome as a target for small regulatory ncRNAs and demonstrate the existence of a yet unknown mechanism of translation regulation. Ribosome-associated ncRNAs (rancRNAs) are found in all domains of life and represent a prevalent but so far largely unexplored class of regulatory molecules (5). Future work on the small ncRNA interactomes of ribosomes in a variety of model systems will allow deeper insight into the conservation and functional repertoire of this emerging class of regulatory ncRNA molecules.

Ribosome-associated ncRNAs (rancRNAs) An emerging class of translation regulators

Small non-protein-coding RNA (ncRNA) molecules represent major contributors to regulatory networks in controlling gene expression in a highly efficient manner. Most of the recently discovered regulatory ncRNAs acting on translation target the mRNA rather than the ribosome (e.g.: miRNAs, siRNAs, antisense RNAs). To address the question, whether ncRNA regulators exist that are capable of modulating the rate of protein production by directly interacting with the ribosome, we have analyzed the small ncRNA interactomes of ribosomes. Deep-sequencing analyses revealed thousands of putative rancRNAs in various model organisms (1,2). For a subset of these ncRNA candidates we have gathered experimental evidence that they associate with ribosomes in a stress-dependent manner and fine-tune the rate of protein biosynthesis (3,4). Many of the investigated rancRNAs appear to be processing products of larger functional RNAs, such as tRNAs (2,3), mRNAs (3), or snoRNAs (2). Post-transcriptional cleavage of RNA to generate smaller fragments is a widespread mechanism that enlarges the structural and functional complexity of cellular RNomes. Our data disclose the ribosome as target for small regulatory RNAs. rancRNAs are found in all domains of life and represent a prevalent but so far largely unexplored class of regulatory molecules (5). Ongoing work in our lab revealed first insight into rancRNA processing and mechanism of this emerging class of translation regulators.

tRNA-derived fragments target the small ribosomal subunit to fine-tune translation

Post-transcriptional cleavage of RNA molecules to generate smaller fragments is a widespread mechanism that enlarges the structural and functional complexity of cellular RNomes. In particular, fragments deriving from both precursor and mature tRNAs represent one of the rapidly growing classes of post-transcriptional RNA pieces. Importantly, these tRNA-derived fragments (tRFs) possess distinct expression patterns, abundance, cellular localizations, or biological roles compared with their parental tRNA molecules [1]. Here we present evidence that tRFs from the archaeon Haloferax volcanii directly bind to ribosomes. In a previous genomic screen for ribosome-associated small RNAs we have identified a 26 residue long fragment originating from the 5’ part of valine tRNA (Val-tRF) to be by far the most abundant tRF in H. volcanii [2]. The Val-tRF is processed in a stress- dependent manner and was found to primarily target the small ribosomal subunit in vitro and in vivo. Translational activity was markedly reduced in the presence of Val-tRF, while control RNA fragments of similar length did not show inhibition of protein biosynthesis. Crosslinking experiments and subsequent primer extension analyses revealed the Val-tRF interaction site to surround the mRNA path in the 30S subunit. In support of this, binding experiments demonstrated that Val-tRF does compete with mRNAs for ribosome binding. Therefore this tRF represents a ribosome-associated non-protein-coding RNA (rancRNA) capable of regulating gene expression in H. volcanii under environmental stress conditions probably by fine-tuning the rate of protein production [3].

tRNA-derived fragments target the small ribosomal subunit to fine-tune translation

Post-transcriptional cleavage of RNA molecules to generate smaller fragments is a widespread mechanism that enlarges the structural and functional complexity of cellular RNomes. In particular, fragments deriving from both precursor and mature tRNAs represent one of the rapidly growing classes of post-transcriptional RNA pieces. Importantly, these tRNA-derived fragments (tRFs) possess distinct expression patterns, abundance, cellular localizations, or biological roles compared with their parental tRNA molecules [1]. Here we present evidence that tRFs from the halophilic archaeon Haloferax volcanii directly bind to ribosomes. In a previous genomic screen for ribosome-associated small RNAs we have identified a 26 residue long fragment originating from the 5’ part of valine tRNA (Val-tRF) to be by far the most abundant tRF in H. volcanii [2]. The Val-tRF is processed in a stress-dependent manner and was found to primarily target the small ribosomal subunit in vitro and in vivo. Translational activity was markedly reduced in the presence of Val-tRF, while control RNA fragments of similar length did not show inhibition of protein biosynthesis. Crosslinking experiments and subsequent primer extension analyses revealed the Val-tRF interaction site to surround the mRNA path in the 30S subunit. In support of this, binding experiments demonstrated that Val-tRF does compete with mRNAs for ribosome binding. Therefore this tRF represents a ribosome-associated non-coding RNA (rancRNA) capable of regulating gene expression in H. volcanii under environmental stress conditions probably by fine-tuning the rate of protein production [3].

NET formation can occur independently of RIPK3 and MLKL signaling

The importance of neutrophil extracellular traps (NETs) in innate immunity is well established but the molecular mechanisms responsible for their formation are still a matter of scientific dispute. Here, we aim to characterize a possible role of the receptor-interacting protein kinase 3 (RIPK3) and the mixed lineage kinase domain-like (MLKL) signaling pathway, which are known to cause necroptosis, in NET formation. Using genetic and pharmacological approaches, we investigated whether this programmed form of necrosis is a prerequisite for NET formation. NETs have been defined as extracellular DNA scaffolds associated with the neutrophil granule protein elastase that are capable of killing bacteria. Neither Ripk3-deficient mouse neutrophils nor human neutrophils in which MLKL had been pharmacologically inactivated, exhibited abnormalities in NET formation upon physiological activation or exposure to low concentrations of PMA. These data indicate that NET formation occurs independently of both RIPK3 and MLKL signaling.

Insulin-like growth factor-I treatment in primary growth hormone insensitivity: effect of recombinant human IGF-I (rhIGF-I) and rhIGF-I/rhIGF-binding protein-3 complex

Growth hormone insensitivity syndrome (GHIS) is a rare cause of growth retardation characterized by high serum GH levels, and low serum insulin-like growth factor I (IGF-I) levels associated with a genetic defect of the GH receptor (GHR) as well post-GHR signaling pathway. Based on clinical, as well as biochemical characteristics, GHIS can be genetically classified as classical/Laron's syndrome and nonclassical/atypical GHIS. Recombinant human IGF-I (rhIGF-I) treatment is effective in promoting growth in subjects who have GHIS. Further, pharmacological studies of a IGF-I compound containing a 1:1 molar complex of rhIGF-I and rhIGF-binding protein-3 (BP-3) demonstrated that the complex was effective in increasing levels of circulating total and free IGF-I and that the administration in patients with GHIS should be safe, well-tolerated and more effective than rhIGF-I on its own.

Epigallocatechin-3-gallate induces cell death in acute myeloid leukaemia cells and supports all-trans retinoic acid-induced neutrophil differentiation via death-associated protein kinase 2

Acute promyelocytic leukaemia (APL) patients are successfully treated with all-trans retinoic acid (ATRA). However, concurrent chemotherapy is still necessary and less toxic therapeutic approaches are needed. Earlier studies suggested that in haematopoietic neoplasms, the green tea polyphenol epigallocatechin-3-gallate (EGCG) induces cell death without adversely affecting healthy cells. We aimed at deciphering the molecular mechanism of EGCG-induced cell death in acute myeloid leukaemia (AML). A significant increase of death-associated protein kinase 2 (DAPK2) levels was found in AML cells upon EGCG treatment paralleled by increased cell death that was significantly reduced upon silencing of DAPK2. Moreover, combined ATRA and EGCG treatment resulted in cooperative DAPK2 induction and potentiated differentiation. EGCG toxicity of primary AML blasts correlated with 67 kDa laminin receptor (67LR) expression. Pretreatment of AML cells with ATRA, causing downregulation of 67LR, rendered these cells resistant to EGCG-mediated cell death. In summary, it was found that (i) DAPK2 is essential for EGCG-induced cell death in AML cells, (ii) ATRA and EGCG cotreatment significantly boosted neutrophil differentiation, and 67LR expression correlates with susceptibility of AML cells to EGCG. We thus suggest that EGCG, by selectively targeting leukaemic cells, may improve differentiation therapies for APL and chemotherapy for other AML subtypes.

Fast kinase domain-containing protein 3 is a mitochondrial protein essential for cellular respiration

Fas-activated serine/threonine phosphoprotein (FAST) is the founding member of the FAST kinase domain-containing protein (FASTKD) family that includes FASTKD1-5. FAST is a sensor of mitochondrial stress that modulates protein translation to promote the survival of cells exposed to adverse conditions. Mutations in FASTKD2 have been linked to a mitochondrial encephalomyopathy that is associated with reduced cytochrome c oxidase activity, an essential component of the mitochondrial electron transport chain. We have confirmed the mitochondrial localization of FASTKD2 and shown that all FASTKD family members are found in mitochondria. Although human and mouse FASTKD1-5 genes are expressed ubiquitously, some of them are most abundantly expressed in mitochondria-enriched tissues. We have found that RNA interference-mediated knockdown of FASTKD3 severely blunts basal and stress-induced mitochondrial oxygen consumption without disrupting the assembly of respiratory chain complexes. Tandem affinity purification reveals that FASTKD3 interacts with components of mitochondrial respiratory and translation machineries. Our results introduce FASTKD3 as an essential component of mitochondrial respiration that may modulate energy balance in cells exposed to adverse conditions by functionally coupling mitochondrial protein synthesis to respiration.

Association of the (CA)n repeat polymorphism of insulin-like growth factor-I and -202 A/C IGF-binding protein-3 promoter polymorphism with adult height in patients with severe growth hormone deficiency

A number of mathematical models for predicting growth and final height outcome have been proposed to enable the clinician to 'individualize' growth-promoting treatment. However, despite optimizing these models, many patients with isolated growth hormone deficiency (IGHD) do not reach their target height. The aim of this study was to analyse the impact of polymorphic genotypes [CA repeat promoter polymorphism of insulin-like growth factor-I (IGF-I) and the -202 A/C promoter polymorphism of IGF-Binding Protein-3 (IGFBP-3)] on variable growth factors as well as final height in severe IGHD following GH treatment. DESIGN, PATIENTS AND CONTROLS: One hundred seventy eight (IGF-I) and 167 (IGFBP-3) subjects with severe growth retardation because of IGHD were studied. In addition, the various genotypes were also studied in a healthy control group of 211 subjects.

Gerbu adjuvant modulates the immune response and thus the course of infection in C56BL/6 mice immunised with Echinococcus multilocularis rec14-3-3 protein

Vaccination with Echinococcus multilocularis 14-3-3 protein can protect mice against primary E. multilocularis infection. The present study investigated the efficacy and efficiency of the adjuvant muramyl dipeptide Gerbu, alone or together with recombinant 14-3-3 protein, to modulate the course of secondary E. multilocularis infection in C56BL/6 mice. The application of Gerbu alone already resulted in a parasite weight reduction when compared with infected control mice, while rec14-3-3 did not add to this effect. Immunological parameters were concurrently assessed with a mixed cell reaction including bone marrow-derived dendritic cells (BMDCs) together with lymph node cells from mice with or without immunisation and/or infection. While mice having received Gerbu adjuvant were found to highly proliferate in response to co-cultivation with 14-3-3-stimulated bone marrow dendritic cells, a sensitisation of BMDCs with vesicle fluid (VF) antigen lead to a striking decrease of the lymphoproliferative response in comparison to that of control mice, raising the hypothesis that immunosuppressive components may be part of this VF-antigen. Anti-14-3-3 antibody production was only found in those mice that had been previously 14-3-3-immunised, whereas all other only-infected mice failed to produce such antibodies. Conclusively, Gerbu adjuvant appears to directly generate a non-specific immune response that contributes to the control of the metacestode growth, putatively in association with a BMDC activity suppressed by components of the VF-antigen.

Gene and protein expression of galectin-3 and galectin-9 in experimental pneumococcal meningitis

Inflammation of the subarachnoid and ventricular space contributes to the development of brain damage i.e. cortical necrosis and hippocampal apoptosis in pneumococcal meningitis (PM). Galectin-3 and -9 are known pro-inflammatory mediators and regulators of apoptosis. Here, the gene and protein expression profile for both galectins was assessed in the disease progression of PM. The mRNA of Lgals3 and Lgals9 increased continuously in the cortex and in the hippocampus from 22 h to 44 h after infection. At 44 h after infection, mRNA levels of Lgals9 in the hippocampus were 7-fold and those of Lgals3 were 30-fold higher than in uninfected controls (P<0.01). Galectin-9 protein did not change, but galectin-3 significantly increased in cortex and hippocampus with the duration of PM. Galectin-3 was localized to polymorphonuclear neutrophils, microglia, monocytes and macrophages, suggesting an involvement of galectin-3 in the neuroinflammatory processes leading to brain damage in PM.