RioK1, a new interactor of protein arginine methyltransferase 5 (PRMT5), competes with pICln for binding and modulates PRMT5 complex composition and substrate specificity

Protein arginine methylation plays a critical role in differential gene expression through modulating protein-protein and protein-DNA/RNA interactions. Although numerous proteins undergo arginine methylation, only limited information is available on how protein arginine methyltransferases (PRMTs) identify their substrates. The human PRMT5 complex consists of PRMT5, WD45/MEP50 (WD repeat domain 45/methylosome protein 50), and pICln and catalyzes the symmetrical arginine dimethylation of its substrate proteins. pICln recruits the spliceosomal Sm proteins to the PRMT5 complex for methylation, which allows their subsequent loading onto snRNA to form small nuclear ribonucleoproteins. To understand how the PRMT5 complex is regulated, we investigated its biochemical composition and identified RioK1 as a novel, stoichiometric component of the PRMT5 complex. We show that RioK1 and pICln bind to PRMT5 in a mutually exclusive fashion. This results in a PRMT5-WD45/MEP50 core structure that either associates with pICln or RioK1 in distinct complexes. Furthermore, we show that RioK1 functions in analogy to pICln as an adapter protein by recruiting the RNA-binding protein nucleolin to the PRMT5 complex for its symmetrical methylation. The exclusive interaction of PRMT5 with either pICln or RioK1 thus provides the first mechanistic insight into how a methyltransferase can distinguish between its substrate proteins.     

Noninvasive online biomass detector system for cultivation in shake flasks

A novel online sensor system for noninvasive and continuous monitoring of cell growth in shake flasks is described. The measurement principle is based on turbidity measurement by detecting 180°‐scattered light and correlation to OD by nonlinear calibration models. The sensor system was integrated into a commercial shaking tablar to read out turbidity from below the shake flasks bottom. The system was evaluated with two model microorganisms, Escherichia coli K12 as prokaryotic and Saccharomyces cerevisiae as eukaryotic model. The sensor allowed an accurate monitoring of turbidity and correlation with OD₆₀₀ ≤ 30. The determination of online OD showed relative errors of about 7.5% for E. coli K12 and 12% for S. cerevisiae. This matches the errors of the laborious offline OD and thus facilitates to overcome the drawbacks of the classical method as risk of contamination and decreasing volumes through sampling. One major challenge was to ensure a defined, nonvarying measurement zone as the rotating suspension in the shake flask forms a liquid sickle which circulates round the flasks inner bottom wall. The resulting alteration of liquid height above the sensor could be compensated by integration of an acceleration sensor into the tablar to synchronize the sensor triggering.     

Efficacy of IFN-λ1 to Protect Human Airway Epithelial Cells against Human Rhinovirus 1B Infection

Impaired interferon (IFN) production has been observed in various obstructive respiratory diseases. This contributes to enhanced sensitivity towards viral infections triggering acute exacerbations. To compensate for this impaired host IFN response, there is need to explore new therapeutic strategies, like exogenous administration of IFNs as prophylactic treatment. In the present study, we examined the protective potential of IFN-λ1 and compared it with the previously established protecting effect of IFN-β. A549 cells and human primary bronchial epithelial cells were first treated with either IFN-β (500 IU/ml) or IFN-λ1 (500 ng/ml) for 18 h. For infection, two approaches were adopted: i) Continuous scenario: after pre-treatment, cells were infected immediately for 24 h with human rhinovirus 1B (HRV1B) in IFN-containing medium, or were cultured for another 72 h in IFN-containing medium, and then infected for 24 h with HRV1B, ii) Pre-treatment scenario: IFN-containing medium was replaced after 18 h and cells were infected for 4 h either immediately after pre-treatment or after additional culturing for 72 h in IFN-free medium. The protective effect was evaluated in terms of reduction in the number of viral copies/infectious progeny, and enhanced expression of IFN-stimulated genes (ISGs). In both cell types and in both approaches, IFN-λ1 and IFN-β treatment resulted in pronounced and long-lasting antiviral effects exemplified by significantly reduced viral copy numbers and diminished infectious progeny. This was associated with strong up-regulation of multiple ISGs. However, in contrast to the IFN-β induced expression of ISGs, which decreased over time, expression of ISGs induced by IFN-λ1 was sustained or even increased over time. Here we demonstrate that the protective potential of IFN-λ1 is comparable to IFN-β. Yet, the long-lasting induction of ISGs by IFN-λ1 and most likely less incitement of side effects due to more localized expression of its receptors could make it an even more promising candidate for prophylactic treatment than IFN-β.     

TPX2 Impacts Acetylation of Histone H4 at Lysine 16: Implications for DNA Damage Response

During interphase, the spindle assembly factor TPX2 is compartmentalized in the nucleus where its roles remain largely uncharacterized. Recently, we found that TPX2 regulates the levels of serine 139-phosphoryated H2AX (γ-H2AX) at chromosomal breaks induced by ionizing radiation. Here, we report that TPX2 readily associates with the chromatin in the absence of ionizing radiation. Overexpression of TPX2 alters the DAPI staining pattern of interphase cells and depletion of TPX2 constitutively decreases the levels of histone H4 acetylated at lysine16 (H4K16ac) during G1-phase. Upon ionizing irradiation, this constitutive TPX2 depletion-dependent decrease in H4K16ac levels correlates with increased levels of γ-H2AX. The inversely correlated levels of H4K16ac and γ-H2AX can also be modified by altering the levels of SIRT1, herein identified as a novel protein complex partner of TPX2. Furthermore, we find that TPX2 depletion also interferes with formation of 53BP1 ionizing radiation-induced foci, known to depend on γ-H2AX and the acetylation status of H4K16. In brief, our study is the first indication of a constitutive control of TPX2 on H4K16ac levels, with potential implications for DNA damage response.     

Deletion of the gliP gene of Aspergillus fumigatus results in loss of gliotoxin production but has no effect on virulence of the fungus in a low-dose mouse infection model

Gliotoxin is a secondary metabolite produced by several fungi including the opportunistic human pathogen Aspergillus fumigatus. As gliotoxin exerts immunosuppressive effects in vitro and in vivo, a role as a virulence determinant in invasive aspergillosis has been discussed for a long time but evidence has not been provided until now. Here, by the use of different selection marker genes A. fumigatus knock-out strains were generated that are deficient for the non-ribosomal peptide synthetase GliP, the putative key enzyme of the gliotoxin biosynthesis. Deletion of the gliP gene resulted in loss of gliotoxin production, as analysed by high performance liquid chromatography and tandem mass spectrometry. No differences in morphology or growth kinetics between wild-type and gliP-deletion strains were observed. In vitro, the culture supernatant of the gliP-deficient strains showed a reduced cytotoxic effect on both macrophage-like cells and T cell lines. In a low-dose murine infection model of invasive aspergillosis, gliotoxin was detected in the lung and absent when mice were infected with the gliP deletion strain. However, gliP deletion strains showed no difference in virulence compared with the corresponding wild-type strains. Taken together, the non-ribosomal peptide synthetase GliP is essential for gliotoxin production in A. fumigatus. Gliotoxin is not required for pathogenicity of the fungus in immunocompromised mice, despite the fact that a reduced cytotoxicity of the culture supernatant of gliP deletion strains was demonstrated.     

Precise centromere mapping using a combination of repeat junction markers and chromatin immunoprecipitation-polymerase chain reaction

Centromeres are difficult to map even in species where genetic resolution is excellent. Here we show that junctions between repeats provide reliable single-copy markers for recombinant inbred mapping within centromeres and pericentromeric heterochromatin. Repeat junction mapping was combined with anti-CENH3-mediated ChIP to provide a definitive map position for maize centromere 8.     

Location of cell cycle regulators cyclin B1, cyclin A, PCNA, Ki67 and cell cycle inhibitors p21, p27 and p57 in human first trimester placenta and deciduas

Although placental development and implantation depend on the coordination of trophoblast proliferation, differentiation and invasion, little is known about the cell cycle regulators that govern the control of these events. The hypothesis that the coordinated expression of cell cycle progression and inhibition factors will determine whether cytotrophoblasts proliferate or undergo cell cycle arrest or cell cycle exit allowing subsequent differentiation was tested. The cell cycle promotors cyclin A, cyclin B1, PCNA, Ki67 and the cell cycle inhibitors p21, p27 and p57 were immunolocalized in tissue sections of first trimester pregnancies (weeks 6 and 9–12). Double staining with cytokeratin 7 allowed unambiguous identification of extravillous cytotrophoblast (EVT) in the decidua. Villous cytotrophoblasts were immunolabelled for Ki67 and cyclin A but only few were stained with anti-cyclin B1. The syncytiotrophoblast was devoid of immunoreactivity for any of the cell cycle progression factors. It expressed especially p21, whereas p27 and p57 were predominantly found in villous cytotrophoblasts. PCNA, Ki67, cyclin A and cyclin B1 were immunolocalized in proximal and distal EVTs of anchoring villi and in EVT which had invaded the upper decidual segments. All EVTs strongly expressed p27 and p57, but not p21. These data clearly suggest different functions for p21, p27 and p57 in placental development with distinct roles for p21 and p57 in syncytiotrophoblast and EVT differentiation, respectively. p27 appears to be involved in both the processes. The results may also challenge the concept of differential mitotic activity in the proximal and distal parts of the first trimester cytotrophoblast cell column, but more functional studies are clearly needed. The presence of p27 and p57 in EVT cells, which invade the deciduas deeply, may account for the loss of mitogenic potential of these cells.     

The Synthetic Antimicrobial Peptide 19-2.5 Interacts with Heparanase and Heparan Sulfate in Murine and Human Sepsis

Heparanase is an endo-β-glucuronidase that cleaves heparan sulfate side chains from their proteoglycans. Thereby, heparanase liberates highly potent circulating heparan sulfate-fragments (HS-fragments) and triggers the fatal and excessive inflammatory response in sepsis. As a potential anti-inflammatory agent for sepsis therapy, peptide 19–2.5 belongs to the class of synthetic anti-lipopolysaccharide peptides; however, its activity is not restricted to Gram-negative bacterial infection. We hypothesized that peptide 19–2.5 interacts with heparanase and/or HS, thereby reducing the levels of circulating HS-fragments in murine and human sepsis. Our data indicate that the treatment of septic mice with peptide 19–2.5 compared to untreated control animals lowers levels of plasma heparanase and circulating HS-fragments and reduces heparanase activity. Additionally, mRNA levels of heparanase in heart, liver, lung, kidney and spleen are downregulated in septic mice treated with peptide 19–2.5 compared to untreated control animals. In humans, plasma heparanase level and activity are elevated in septic shock. The ex vivo addition of peptide 19–2.5 to plasma of septic shock patients decreases heparanase activity but not heparanase level. Isothermal titration calorimetry revealed a strong exothermic reaction between peptide 19–2.5 and heparanase and HS-fragments. However, a saturation character has been identified only in the peptide 19–2.5 and HS interaction. In conclusion, the findings of our current study indicate that peptide 19–2.5 interacts with heparanase, which is elevated in murine and human sepsis and consecutively attenuates the generation of circulating HS-fragments in systemic inflammation. Thus, peptide 19–2.5 seems to be a potential anti-inflammatory agent in sepsis.     

Compartmentation of phenylacetic and cinnamic acid synthesis in spinach

Applying labelled phenylalanine or tyrosine to purified intact spinach chloroplasts, only the corresponding phenylacetic acids but not the cinnamic acids could be detected. The addition of mercaptoethanol or dl -dithiothreitol and the variation of light conditions had only a slight effect. However, cinnamic acids could be found together with phenylacetic acids in leaf homogenates indicating the presence of phenylalanine and/or tyrosine ammonia lyase outside the spinach chloroplasts. Similar results were obtained with barley leaf homogenates, where cinnamic acids were the main products. Reviewing recent findings on amino acid synthesis in spinach leaves, it may be concluded that the synthesis of aromatic amino acids is restricted to the chloroplast, whereas the metabolism of secondary aromatic compounds is predominantly localized outside the chloroplasts.