A proteomic view of the facultatively chemolithoautotrophic lifestyle of Ralstonia eutropha H16

Ralstonia eutropha H16 is an H₂‐oxidizing, facultative chemolithoautotroph. Using 2‐DE in conjunction with peptide mass spectrometry we have cataloged the soluble proteins of this bacterium during growth on different substrates: (i) H₂ and CO₂, (ii) succinate and (iii) glycerol. The first and second conditions represent purely lithoautotrophic and purely organoheterotrophic nutrition, respectively. The third growth regime permits formation of the H₂‐oxidizing and CO₂‐fixing systems concomitant to utilization of an organic substrate, thus enabling mixotrophic growth. The latter type of nutrition is probably the relevant one with respect to the situation faced by the organism in its natural habitats, i.e. soil and mud. Aside from the hydrogenase and Calvin‐cycle enzymes, the protein inventories of the H₂‐CO₂‐ and succinate‐grown cells did not reveal major qualitative differences. The protein complement of the glycerol‐grown cells resembled that of the lithoautotrophic cells. Phosphoenolpyruvate (PEP) carboxykinase was present under all three growth conditions, whereas PEP carboxylase was not detectable, supporting earlier findings that PEP carboxykinase is alone responsible for the anaplerotic production of oxaloacetate from PEP. The elevated levels of oxidative stress proteins in the glycerol‐grown cells point to a significant challenge by ROS under these conditions. The results reported here are in agreement with earlier physiological and enzymological studies indicating that R. eutropha H16 has a heterotrophic core metabolism onto which the functions of lithoautotrophy have been grafted.     

Ultrastructure,tactic behaviour and potential for sulfate reduction of a novel multicellular magnetotactic prokaryote from North Sea sediments

Multicellular magnetotactic prokaryotes (MMPs) represent highly organized, spherical and motile aggregates of 10–40 bacterial cells containing magnetosomes. Although consisting of different cells, each with its own magnetosomes and flagellation, MMPs orient themselves within a magnetic field and exhibit magnetotaxis. So far, MMPs have only been found in several North and South American coastal lagoons and salt marshes. In the present study, a novel type of MMP was discovered in coastal tidal sand flats of the North Sea. High‐resolution scanning electron microscopy revealed the presence of bullet‐shaped magnetosomes which were aligned in several parallel chains. Within each aggregate, the magnetosome chains of individual cells were oriented in the same direction. Energy dispersive X‐ray (EDX) analysis showed that the magnetosomes are composed of iron sulfide. This particular morphology and arrangement of magnetosomes has previously not been reported for other MMPs. 16S rRNA gene sequence analysis revealed a single phylotype which represented a novel phylogenetic lineage with ≥ 4% sequence divergence to all previously described MMP sequences and was related to the dissimilatory sulfate‐reducing Desulfosarcina variabilis within the family Desulfobacteraceae of the subphylum Deltaproteobacteria. Fluorescence in situ hybridization with a specific oligonucleotide probe revealed that all MMPs in the tidal flat sediments studied belonged to the novel phylotype. Within each MMP, all bacterial cells showed a hybridization signal, indicating that the aggregates are composed of cells of the same phylotype. Genes for dissimilatory sulfite reductase (dsrAB) and dissimilatory adenosine‐5′‐phosphate reductase (aprA) could be detected in purified MMP samples, suggesting that MMPs are capable of sulfate reduction. Chemotaxis assays with 41 different test compounds yielded strong responses towards acetate and propionate, whereas other organic acids, alcohols, sugars, sugar alcohols or sulfide did not elicit any response. By means of its coordinated magnetotaxis and chemotaxis, the novel type of MMP is well adapted to the steep chemical gradients which are characteristic for intertidal marine sediments.     

Chemical Analysis and Risk Assessment of Diethyl Phthalate in Alcoholic Beverages with Special Regard to Unrecorded Alcohol

Background

Phthalates are synthetic compounds with a widespread field of applications. For example, they are used as plasticizers in PVC plastics and food packaging, or are added to personal care products. Diethyl phthalate (DEP) may be used to denature alcohol, e.g., for cosmetic purposes. Public health concerns of phthalates include carcinogenic, teratogenic, hepatotoxic and endocrine effects. The aim of this study was to develop and validate a method for determining phthalates in alcohol samples and to provide a risk assessment for consumers of such products.

Methodology/Principal Findings

A liquid-liquid extraction procedure was optimized by varying the following parameters: type of extraction solvent (cyclohexane, n-hexane, 1,1,2-trichlorotrifluoroethane), the ratio extraction solvent/sample volume (1∶1 to 50∶1) and the number of extraction repetitions (1–10). The best extraction yield (99.9%) was achieved with the solvent 1,1,2-trichlorotrifluoroethane, an extraction solvent volume/sample volume ratio of 10∶1 and a double extraction. For quantification, gas chromatography/mass spectrometry with deuterated internal standards was used. The investigated samples were alcoholic beverages and unrecorded alcohol products from different countries (n = 257). Two unrecorded alcohol samples from Lithuania contained diethyl phthalate in concentrations of 608 mg/L and 210 mg/L.

Conclusions/Significance

The consumption of the phthalate-positive unrecorded alcohols would exceed tolerable daily intakes as derived from animal experiments. Both positive samples were labelled as cosmetic alcohol, but had clearly been offered for human consumption. DEP seems to be unsuitable as a denaturing agent as it has no effect on the organoleptic properties of ethanol. In light of our results that DEP might be consumed by humans in unrecorded alcohols, the prohibition of its use as a denaturing agent should be considered.     

Inherited Glutathione Reductase Deficiency and Plasmodium falciparum Malaria—A Case Study

In Plasmodium falciparum-infected red blood cells (RBCs), the flavoenzyme glutathione reductase (GR) regenerates reduced glutathione, which is essential for antioxidant defense. GR utilizes NADPH produced in the pentose phosphate shunt by glucose-6-phosphate dehydrogenase (G6PD). Thus, conditions affecting host G6PD or GR induce increased sensitivity to oxidants. Hereditary G6PD deficiency is frequent in malaria endemic areas and provides protection against severe malaria. Furthermore, GR deficiency resulting from insufficient saturation of the enzyme with its prosthetic group FAD is common. Based on these naturally occurring phenomena, GR of malaria parasites and their host cells represent attractive antimalarial drug targets. Recently we were given the opportunity to examine invasion, growth, and drug sensitivity of three P. falciparum strains (3D7, K1, and Palo Alto) in the RBCs from three homozygous individuals with total GR deficiency resulting from mutations in the apoprotein. Invasion or growth in the GR-deficient RBCs was not impaired for any of the parasite strains tested. Drug sensitivity to chloroquine, artemisinin, and methylene blue was comparable to parasites grown in GR-sufficient RBCs and sensitivity towards paraquat and sodium nitroprusside was only slightly enhanced. In contrast, membrane deposition of hemichromes as well as the opsonizing complement C3b fragments and phagocytosis were strongly increased in ring-infected RBCs of the GR-deficient individuals compared to ring-infected normal RBCs. Also, in one of the individuals, membrane-bound autologous IgGs were significantly enhanced. Thus, based on our in vitro data, GR deficiency and drug-induced GR inhibition may protect from malaria by inducing enhanced ring stage phagocytosis rather than by impairing parasite growth directly.     

Bacterial Porin Disrupts Mitochondrial Membrane Potential and Sensitizes Host Cells to Apoptosis

The bacterial PorB porin, an ATP-binding β-barrel protein of pathogenic Neisseria gonorrhoeae, triggers host cell apoptosis by an unknown mechanism. PorB is targeted to and imported by host cell mitochondria, causing the breakdown of the mitochondrial membrane potential (ΔΨ_m). Here, we show that PorB induces the condensation of the mitochondrial matrix and the loss of cristae structures, sensitizing cells to the induction of apoptosis via signaling pathways activated by BH3-only proteins. PorB is imported into mitochondria through the general translocase TOM but, unexpectedly, is not recognized by the SAM sorting machinery, usually required for the assembly of β-barrel proteins in the mitochondrial outer membrane. PorB integrates into the mitochondrial inner membrane, leading to the breakdown of ΔΨ_m. The PorB channel is regulated by nucleotides and an isogenic PorB mutant defective in ATP-binding failed to induce ΔΨ_m loss and apoptosis, demonstrating that dissipation of ΔΨ_m is a requirement for cell death caused by neisserial infection.     

Growth Efficiency and Carbon Balance for the Sponge Haliclona oculata

To obtain more knowledge about carbon requirements for growth by sponges, the growth rate, respiration rate, and clearance rate was measured in situ in Haliclona oculata. We found that only 34% of the particulate carbon pumped through the sponge was used for both respiration and growth. The net growth efficiency, being the ratio of carbon incorporated in biomass and the total carbon used by the sponge for respiration and growth, was found to be 0.099 ± 0.013. Thus, about 10% of the total used carbon was fixed in biomass, and over 90% was used for generating energy for growth, maintenance, reproduction, and pumping. H. oculata had 2.5 μmol C available for every micromole O₂ consumed. A value of 0.75 for respiratory quotient (RQ in micromole CO₂ micromole O₂⁻¹) was used for H. oculata, which is the average value reported in literature for different marine invertebrates. Thus, carbon was available in excess to meet the respiratory demand. Oxygen was found not to be the limiting factor for growth, since only 3.3% of the oxygen pumped through the sponge body was used. Our results indicate that both oxygen and carbon availability are not limiting. The low growth efficiency agrees with the low growth rates found for the species used in this study.     

DDX6 recruits translational silenced human reticulocyte 15-lipoxygenase mRNA to RNP granules

Erythroid precursor cells lose the capacity for mRNA synthesis due to exclusion of the nucleus during maturation. Therefore, the stability and translation of mRNAs that code for specific proteins, which function in late stages of maturation when reticulocytes become erythrocytes, are controlled tightly. Reticulocyte 15-lipoxygenase (r15-LOX) initiates the breakdown of mitochondria in mature reticulocytes. Through the temporal restriction of mRNA translation, the synthesis of r15-LOX is prevented in premature cells. The enzyme is synthesized only in mature reticulocytes, although r15-LOX mRNA is already present in erythroid precursor cells. Translation of r15-LOX mRNA is inhibited by hnRNP K and hnRNP E1, which bind to the differentiation control element (DICE) in its 3' untranslated region (3'UTR). The hnRNP K/E1-DICE complex interferes with the joining of the 60S ribosomal subunit to the 40S subunit at the AUG. We took advantage of the inducible human erythroid K562 cell system that fully recapitulates this process to identify so far unknown factors, which are critical for DICE-dependent translational regulation. Applying RNA chromatography with the DICE as bait combined with hnRNP K immunoprecipitation, we specifically purified the DEAD-box RNA helicase 6 (DDX6) that interacts with hnRNP K and hnRNP E1 in a DICE-dependent manner. Employing RNA interference and fluorescence in situ hybridization, we show that DDX6 colocalizes with endogenous human (h)r15-LOX mRNA to P-body-like RNP granules, from which 60S ribosomal subunits are excluded. Our data suggest that in premature erythroid cells translational silencing of hr15-LOX mRNA is maintained by DDX6 mediated storage in these RNP granules.     

Biomarker discovery in heterogeneous tissue samples -taking the in-silico deconfounding approach

Background  

For heterogeneous tissues, such as blood, measurements of gene expression are confounded by relative proportions of cell types involved. Conclusions have to rely on estimation of gene expression signals for homogeneous cell populations, e.g. by applying micro-dissection, fluorescence activated cell sorting, or in-silico deconfounding. We studied feasibility and validity of a non-negative matrix decomposition algorithm using experimental gene expression data for blood and sorted cells from the same donor samples. Our objective was to optimize the algorithm regarding detection of differentially expressed genes and to enable its use for classification in the difficult scenario of reversely regulated genes. This would be of importance for the identification of candidate biomarkers in heterogeneous tissues.     

3-Substituted gem-cyclohexane sulfone based gamma-secretase inhibitors for Alzheimer's disease: conformational analysis and biological activity

Previously, chemistry effort on the gem-cyclohexane series of gamma-secretase inhibitors has focused on the 4-position of the cyclohexane ring. Recently chemistry has been directed towards the 3-position and substitution here has also provided compounds with high gamma-secretase activity.