High-resolution structure of the histidine-containing phosphocarrier protein (HPr) from Staphylococcus aureus and characterization of its interaction with the bifunctional HPr kinase/phosphorylase

A high-resolution structure of the histidine-containing phosphocarrier protein (HPr) from Staphylococcus aureus was obtained by heteronuclear multidimensional nuclear magnetic resonance (NMR) spectroscopy on the basis of 1,766 structural restraints. Twenty-three hydrogen bonds in HPr could be directly detected by polarization transfer from the amide nitrogen to the carbonyl carbon involved in the hydrogen bond. Differential line broadening was used to characterize the interaction of HPr with the HPr kinase/phosphorylase (HPrK/P) of Staphylococcus xylosus, which is responsible for phosphorylation-dephosphorylation of the hydroxyl group of the regulatory serine residue at position 46. The dissociation constant Kd was determined to be 0.10 +/- 0.02 mM at 303 K from the NMR data, assuming independent binding. The data are consistent with a stoichiometry of 1 HPr molecule per HPrK/P monomer in solution. Using transversal relaxation optimized spectroscopy-heteronuclear single quantum correlation, we mapped the interaction site of the two proteins in the 330-kDa complex. As expected, it covers the region around Ser46 and the small helix b following this residue. In addition, HPrK/P also binds to the second phosphorylation site of HPr at position 15. This interaction may be essential for the recognition of the phosphorylation state of His15 and the phosphorylation-dependent regulation of the kinase/phosphorylase activity. In accordance with this observation, the recently published X-ray structure of the HPr/HPrK core protein complex from Lactobacillus casei shows interactions with the two phosphorylation sites. However, the NMR data also suggest differences for the full-length protein from S. xylosus: there are no indications for an interaction with the residues preceding the regulatory Ser46 residue (Thr41 to Lys45) in the protein of S. xylosus. In contrast, it seems to interact with the C-terminal helix of HPr in solution, an interaction which is not observed for the complex of HPr with the core of HPrK/P of L. casei in crystals.     

Free fatty acid palmitate impairs the vitality and function of cultured human bladder smooth muscle cells

Background

Incidence of urinary tract infections is elevated in patients with diabetes mellitus. Those patients show increased levels of the saturated free fatty acid palmitate. As recently shown metabolic alterations induced by palmitate include production and secretion of the pro-inflammatory cytokine interleukine-6 (IL-6) in cultured human bladder smooth muscle cells (hBSMC). Here we studied the influence of palmitate on vital cell properties, for example, regulation of cell proliferation, mitochondrial enzyme activity and antioxidant capacity in hBSMC, and analyzed the involvement of major cytokine signaling pathways.

Methodology/Principal Findings

HBSMC cultures were set up from bladder tissue of patients undergoing cystectomy and stimulated with palmitate. We analyzed cell proliferation, mitochondrial enzyme activity, and antioxidant capacity by ELISA and confocal immunofluorescence. In signal transduction inhibition experiments we evaluated the involvement of NF-κB, JAK/STAT, MEK1, PI3K, and JNK in major cytokine signaling pathway regulation. We found: (i) palmitate decreased cell proliferation, increased mitochondrial enzyme activity and antioxidant capacity; (ii) direct inhibition of cytokine receptor by AG490 even more strongly suppressed cell proliferation in palmitate-stimulated cells, while counteracting palmitate-induced increase of antioxidant capacity; (iii) in contrast knockdown of the STAT3 inhibitor SOCS3 increased cell proliferation and antioxidant capacity; (iv) further downstream JAK/STAT3 signaling cascade the inhibition of PI3K or JNK enhanced palmitate induced suppression of cell proliferation; (v) increase of mitochondrial enzyme activity by palmitate was enhanced by inhibition of PI3K but counteracted by inhibition of MEK1.

Conclusions/Significance

Saturated free fatty acids (e.g., palmitate) cause massive alterations in vital cell functions of cultured hBSMC involving distinct major cytokine signaling pathways. Thereby, certain cytokines might counteract the palmitate-induced downregulation of cell proliferation and vitality. This could be an important link to clinical findings of increased risk of metabolic related bladder diseases such as overactive bladder (OAB) and bladder pain syndrome/interstitial cystitis (BPS/IC).     

Environmental impacts of decommissioning nuclear power plants: methodical challenges,case study,and implications

Purpose

Environmental impacts of the decommissioning of nuclear power plants are brought into focus by the nuclear phase-out in Germany and a worldwide growing number of decommissioning projects. So far, life cycle assessments of decommissioning nuclear power plants have been conducted very rarely or are based on rather uncertain assumptions. Against this background, environmental impacts of the ongoing decommissioning of the nuclear power plant in Lubmin (KGR), Germany are examined. Methodological aspects like transferability to other decommissioning projects as well as influence of assumptions about the lifespan of a power plant are discussed.

Methods

A life cycle assessment of the decommissioning according to ISO 14040/44 is conducted. The decommissioning of one power plant (of the assessed KGR) is chosen as functional unit. The system boundaries include removal and demolition of plant components and buildings as well as decontamination, conditioning, interim storage, and final repository of low-level and interim-level nuclear waste together with disposal and recycling of conventional waste. Interim storage and final repository of high-level nuclear waste such as fuel rods are excluded from the system boundaries as they are assigned to the use phase of the plant. Primary data was obtained from the plant decommissioning firm (Energiewerke Nord GmbH, EWN) in Lubmin. The GaBi database was used to model background processes. Environmental impacts are estimated using the CML2001 methodology.

Results and discussion

Environmental impacts are mainly caused by on-site energetic demands of component removal and peripheral tasks. Further significant impacts are caused by the handling, storage, and final repository of low-level and intermediate-level nuclear waste. Recycling conventional, nonradioactive metallic waste has the potential to unburden the process in a significant scale, depending on recycling rates.

Conclusions

The dismantling of nuclear power plants shows a relevant environmental impact. Regarding the environmental impacts per kilowatt-hour assumptions concerning the plant’ lifespan are a crucial factor. Comparing the result from this study to recent datasets for nuclear power poses the question if LCA datasets represent environmental burdens of nuclear power accurately. The transferability of LCA results to other studies using one parameter for scaling is problematic and needs further research.     

The nitrite-oxidizing community in activated sludge from a municipal wastewater treatment plant determined by fatty acid methyl ester-stable isotope probing

Metabolically-active autotrophic nitrite oxidizers from activated sludge were labeled with ¹³C-bicarbonate under exposure to different temperatures and nitrite concentrations. The labeled samples were characterized by FAME-SIP (fatty acid methyl ester-stable isotope probing). The compound cis-11-palmitoleic acid, which is the major lipid of the most abundant nitrite oxidizer in activated sludge, Candidatus Nitrospira defluvii, showed ¹³C-incorporation in all samples exposed to 3 mM nitrite. Subsequently, the lipid cis-7-palmitoleic acid was labeled, and it indicated the activity of a nitrite oxidizer that was different from the known Nitrospira taxa in activated sludge. The highest incorporation of cis-7-palmitoleic acid label was found after incubation with a nitrite concentration of 0.3 mM at 17 and 22 °C. While activity of Nitrobacter populations could not be detected by the FAME-SIP approach, an unknown nitrite oxidizer with the major lipid cis-9 isomer of palmitoleic acid exhibited ¹³C-incorporation at 28 °C with 30 mM nitrite. These results indicated flexibility of nitrite-oxidizing guilds in a complex community responding to different conditions. Labeled lipids so far not described for activated sludge-associated nitrifiers indicated the presence of unknown nitrite oxidizers in this habitat. The FAME-SIP-based information can be used to define appropriate conditions for the enrichment of nitrite-oxidizing guilds from complex samples.     

Flow cytometric determination of E-selectin, vascular cell adhesion molecule-1, and intercellular cell adhesion molecule-1 in formaldehyde-fixed endothelial cell monolayers

BACKGROUND: Endothelial cell adhesion molecules are involved in initiation and progression of vascular diseases. The purpose of this study was to determine conditions of fixation and dissociation of human umbilical vein endothelial cell (HUVEC) monolayers that permit a reliable flow cytometric determination of intracellular and surface content of E-selectin, vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1). METHODS: TNFalpha-treated HUVEC monolayers were fixed with 0.5% formaldehyde at the end of the experimental incubation. Subsequently, either the monolayer was trypsinized and thereafter the cells were subjected to indirect fluorescence labeling or the monolayer was first labeled and then dissociated by trypsinization. Cell integrity was assessed by vimentin staining. Total adhesion molecule content was detected in saponin-permeabilized cells. RESULTS: HUVEC integrity was maintained when the fixation time of the monolayer did not exceed 5 min and trypsin/EDTA was used for dissociation. Surface adhesion molecules were partially hydrolyzed by trypsin when trypsinization preceded labeling but antibody binding protected adhesion molecules from degradation. VCAM-1 and E-selectin exhibited substantial trypsin-sensitive surface fractions but surface ICAM-1 was mainly trypsin resistant. Permeabilization with 0.06% saponin allowed the detection of considerable intracellular pools of the investigated adhesion molecules. CONCLUSIONS: The described method permits the reliable determination of surface and intracellular fractions of adhesion molecules in formaldehyde-fixed HUVEC monolayers and may be used for studies on the regulation of adhesion molecule expression.     

Web-accessible proteome databases for microbial research

The analysis of proteomes of biological organisms represents a major challenge of the post-genome era. Classical proteomics combines two-dimensional electrophoresis (2-DE) and mass spectrometry (MS) for the identification of proteins. Novel technologies such as isotope coded affinity tag (ICAT)-liquid chromatography/mass spectrometry (LC/MS) open new insights into protein alterations. The vast amount and diverse types of proteomic data require adequate web-accessible computational and database technologies for storage, integration, dissemination, analysis and visualization. A proteome database system (http://www.mpiib-berlin.mpg.de/2D-PAGE) for microbial research has been constructed which integrates 2-DE/MS, ICAT-LC/MS and functional classification data of proteins with genomic, metabolic and other biological knowledge sources. The two-dimensional polyacrylamide gel electrophoresis database delivers experimental data on microbial proteins including mass spectra for the validation of protein identification. The ICAT-LC/MS database comprises experimental data for protein alterations of mycobacterial strains BCG vs. H37Rv. By formulating complex queries within a functional protein classification database "FUNC_CLASS" for Mycobacterium tuberculosis and Helicobacter pylori the researcher can gather precise information on genes, proteins, protein classes and metabolic pathways. The use of the R language in the database architecture allows high-level data analysis and visualization to be performed "on-the-fly". The database system is centrally administrated, and investigators without specific bioinformatic competence in database construction can submit their data. The database system also serves as a template for a prototype of a European Proteome Database of Pathogenic Bacteria. Currently, the database system includes proteome information for six strains of microorganisms.     

Association of Bcl-2 with misfolded prion protein is linked to the toxic potential of cytosolic PrP

Protein misfolding is linked to different neurodegenerative disorders like Alzheimer's disease, polyglutamine, and prion diseases. We investigated the cytotoxic effects of aberrant conformers of the prion protein (PrP) and show that toxicity is specifically linked to misfolding of PrP in the cytosolic compartment and involves binding of PrP to the anti-apoptotic protein Bcl-2. PrP targeted to different cellular compartments, including the cytosol, nucleus, and mitochondria, adopted a misfolded and partially proteinase K-resistant conformation. However, only in the cytosol did the accumulation of misfolded PrP induce apoptosis. Apoptotic cell death was also induced by two pathogenic mutants of PrP, which are partially localized in the cytosol. A mechanistic analysis revealed that the toxic potential is linked to an internal domain of PrP (amino acids 115-156) and involves coaggregation of cytosolic PrP with Bcl-2. Increased expression of the chaperones Hsp70 and Hsp40 prevented the formation of PrP/Bcl-2 coaggregates and interfered with PrP-induced apoptosis. Our study reveals a compartment-specific toxicity of PrP misfolding that involves coaggregation of Bcl-2 and indicates a protective role of molecular chaperones.     

Induction,rapid fixation and retention of mutations in vegetatively propagated banana

Mutation discovery technologies have enabled the development of reverse genetics for many plant species and allowed sophisticated evaluation of the consequences of mutagenesis. Such methods are relatively straightforward for seed‐propagated plants. To develop a platform suitable for vegetatively propagated species, we treated isolated banana shoot apical meristems with the chemical mutagen ethyl methanesulphonate, recovered plantlets and screened for induced mutations. A high density of GC‐AT transition mutations were recovered, similar to that reported in seed‐propagated polyploids. Through analysis of the inheritance of mutations, we observed that genotypically heterogeneous stem cells resulting from mutagenic treatment are rapidly sorted to fix a single genotype in the meristem. Further, mutant genotypes are stably inherited in subsequent generations. Evaluation of natural nucleotide variation showed the accumulation of potentially deleterious heterozygous alleles, suggesting that mutation induction may uncover recessive traits. This work therefore provides genotypic insights into the fate of totipotent cells after mutagenesis and suggests rapid approaches for mutation‐based functional genomics and improvement of vegetatively propagated crops.