Role of proinflammatory markers and NT-proBNP in patients with an implantable cardioverter-defibrillator and an electrical storm

BackgroundSeveral studies have attempted to identify risk factors for the development of an electrical storm (ES), which is defined as ⩾3 separate ventricular tachyarrhythmic (VT/VF) events, but in the majority of studies no triggers have been found. However, little is known about the role of inflammation and NT-proBNP in patients with ES. The aim of this study was therefore to assess the relationship of Interleukin-6 (IL-6), high-sensitivity C-reactive protein (hs-CRP) and NT-proBNP serum concentrations in ICD-patients with or without single spontaneous ventricular tachyarrhythmic events (VT/VF) and in ES.MethodsMarkers were determined in 51 patients without ICD-intervention, in 15 ICD-patients with single VT/VF-episodes during 9-months follow-up and in 20 ICD-patients with ES (blood sampling performed within 60 min after fulfilling ES criteria). VT/VF-episodes were analysed by stored ICD-electrograms.ResultsAll patients had idiopathic dilated cardiomyopathy (n = 23) or coronary artery disease (n = 63). Patients with ES revealed significantly higher mean serum concentrations of all markers (IL-6 15.19 ± 10.34 pg/mL, hs-CRP 20.12 ± 14.4 mg/L, NT-proBNP 4799 ± 4596 pg/mL) compared to baseline values of patients with single VT/VF-events during follow-up (IL-6 8.37 ± 5.8 pg/mL (p = 0.03), hs-CRP 4.7 ± 5.3 mg/dL (p < 0.001), NT-proBNP 1913 ± 2665 pg/mL (p = 0.04)) and compared to baseline values of ICD-patients without device intervention (IL-6 4.62 ± 3.66 pg/mL (p < 0.001), hs-CRP 4.1 ± 3.4 mg/L (p < 0.001), NT-proBNP 1461 ± 2281 pg/mL (p < 0.001)). In 9/20 patients presenting with ES (45%) baseline values were available. All markers were significantly higher during ES compared to event-free determination (IL-6 14.54 ± 10.43 vs. 7.03 ± 2.83 pg/mL (p = 0.04), hs-CRP 19.07 ± 16.07 vs. 6.5 ± 3.9 mg/L (p = 0.02), NT-proBNP 4218 ± 2561 vs. 2099 ± 1279 pg/mL (p = 0.03)).ConclusionsElectrical storm is associated with significantly elevated IL-6, hs-CRP and NT-proBNP serum concentrations in ICD-patients with structural heart disease. Thus, ES may be triggered by proinflammatory activity. Combined intraindividual elevation of determined markers might help to identify patients at risk of impending electrical storm.     

Extracellular Iron Biomineralization by Photoautotrophic Iron-Oxidizing Bacteria

Iron oxidation at neutral pH by the phototrophic anaerobic iron-oxidizing bacterium Rhodobacter sp. strain SW2 leads to the formation of iron-rich minerals. These minerals consist mainly of nano-goethite (α-FeOOH), which precipitates exclusively outside cells, mostly on polymer fibers emerging from the cells. Scanning transmission X-ray microscopy analyses performed at the C K-edge suggest that these fibers are composed of a mixture of lipids and polysaccharides or of lipopolysaccharides. The iron and the organic carbon contents of these fibers are linearly correlated at the 25-nm scale, which in addition to their texture suggests that these fibers act as a template for mineral precipitation, followed by limited crystal growth. Moreover, we evidence a gradient of the iron oxidation state along the mineralized fibers at the submicrometer scale. Fe minerals on these fibers contain a higher proportion of Fe(III) at cell contact, and the proportion of Fe(II) increases at a distance from the cells. All together, these results demonstrate the primordial role of organic polymers in iron biomineralization and provide first evidence for the existence of a redox gradient around these nonencrusting, Fe-oxidizing bacteria.Fe(II) can serve as a source of electrons for phylogenetically diverse microorganisms that precipitate iron minerals as products of their metabolism (see, e.g., references 3, 5, 25, and 30). For example, mixotrophic or autotrophic bacteria can couple the oxidation of Fe(II) to the reduction of nitrate in anoxic and neutral-pH environments. With Fe(III) being highly insoluble at neutral pH, this metabolism leads to the formation of poorly to well-crystallized iron minerals (3, 18, 26, 27) that precipitate partly within the cell periplasm for some strains (22). Similar Fe minerals are also synthesized by autotrophic bacteria that perform anoxygenic photosynthesis, using Fe(II) as an electron donor and light as a source of energy for CO₂ fixation (8, 12, 30), according to the equation HCO₃⁻ + 4 Fe²⁺ + 10 H₂O ⇆ <CH₂O> + 4 Fe(OH)₃ + 7 H⁺.However, the biological mechanisms of iron oxidation in these bacteria and in particular the way they cope with the formation of minerals within their ultrastructures are still not fully understood. Indeed, iron minerals are potentially lethal since their precipitation may alter cellular ultrastructures but also catalyze the production of free radicals (2). Recent genetic studies of the phototrophic, iron-oxidizing bacteria Rhodobacter sp. strain SW2 (6) and Rhodopseudomonas palustris strain TIE-1 (16) have identified genes (fox and pio operons, respectively) encoding proteins specific for iron oxidation. Interestingly, Jiao and Newman (16) suggested that one of these proteins could have a periplasmic localization. However, in contrast to what has been observed in some other phototrophic iron oxidizers (25) and in some nitrate-reducing, iron-oxidizing bacteria (22), no iron-mineral precipitation occurs within the periplasm of the purple nonsulfur iron-oxidizing bacterium Rhodobacter sp. strain SW2 (3). Similarly to some other anaerobic neutrophilic (22, 25) and microaerobic iron-oxidizing bacteria (5, 10), this strain seems indeed to have the ability to localize iron biomineralization at a distance from the cells, leaving large areas of the cells free of precipitates (17, 25). While it has been shown that the Gallionella and Leptothrix genera, for example, produce extracellular polymers that facilitate the nucleation of iron minerals outside cells (see, e.g., references 5 and 9), only a little is known about the existence and function of such polymers in anaerobic, neutrophilic iron-oxidizing bacteria and particularly in the phototrophic strain SW2. In the present study, we investigate iron biomineralization by the photoautotrophic iron-oxidizing bacterium Rhodobacter sp. strain SW2. We use scanning transmission X-ray microscopy (STXM) to map and identify organic polymers produced by the cells as well as the redox state of iron at the 25-nanometer scale regularly during a 2 week-period. These results demonstrate the primordial role of organic polymers in iron biomineralization and provide the first evidence for the existence of a redox gradient around SW2 cells.     

Glutamate receptor properties of human mesencephalic neural progenitor cells: NMDA enhances dopaminergic neurogenesis in vitro

Human midbrain‐derived neural progenitor cells (NPCs) may serve as a continuous source of dopaminergic neurons for the development of novel regenerative therapies in Parkinson’s disease. However, the molecular and functional characteristics of glutamate receptors in human NPCs are largely unknown. Here, we show that differentiated human mesencepahlic NPCs display a distinct pattern of glutamate receptors. In whole‐cell patch‐clamp recordings, l ‐glutamate and NMDA elicited currents in 93% of NPCs after 3 weeks of differentiation in vitro. The concentration‐response plots of differentiated NPCs yielded an EC₅₀ of 2.2 μM for glutamate and an EC₅₀ of 36 μM for NMDA. Glutamate‐induced currents were markedly inhibited by memantine in contrast to 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) suggesting a higher density of functional NMDA than alpha‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA)/kainate receptors. NMDA‐evoked currents and calcium signals were blocked by the NR2B‐subunit specific antagonist ifenprodil indicating functional expression of NMDA receptors containing subunits NR1 and NR2B. In calcium imaging experiments, the blockade of voltage‐gated calcium channels by verapamil abolished AMPA‐induced calcium responses but only partially reduced NMDA‐evoked transients suggesting the expression of calcium‐impermeable, GluR2‐containing AMPA receptors. Quantitative real‐time PCR showed a predominant expression of subunits NR2A and NR2B (NMDA), GluR2 (AMPA), GluR7 (kainate), and mGluR3 (metabotropic glutamate receptor). Treatment of NPCs with 100 μM NMDA in vitro during proliferation (2 weeks) and differentiation (1 week) increased the amount of tyrosine hydroxylase‐immunopositive cells significantly, which was reversed by addition of memantine. These data suggest that NMDA receptors in differentiating human mesencephalic NPCs are important regulators of dopaminergic neurogenesis in vitro.     

The polysialic acid-specific O-acetyltransferase OatC from Neisseria meningitidis serogroup C evolved apart from other bacterial sialate O-acetyltransferases

Neisseria meningitidis serogroup C is a major cause of bacterial meningitis and septicaemia. This human pathogen is protected by a capsule composed of alpha2,9-linked polysialic acid that represents an important virulence factor. In the majority of strains, the capsular polysaccharide is modified by O-acetylation at C-7 or C-8 of the sialic acid residues. The gene encoding the capsule modifying O-acetyltransferase is part of the capsule gene complex and shares no sequence similarities with other proteins. Here, we describe the purification and biochemical characterization of recombinant OatC. The enzyme was found as a homodimer, with the first 34 amino acids forming an efficient oligomerization domain that worked even in a different protein context. Using acetyl-CoA as donor substrate, OatC transferred acetyl groups exclusively onto polysialic acid joined by alpha2,9-linkages and did not act on free or CMP-activated sialic acid. Motif scanning revealed a nucleophile elbow motif (GXS286XGG), which is a hallmark of alpha/beta-hydrolase fold enzymes. In a comprehensive site-directed mutagenesis study, we identified a catalytic triad composed of Ser-286, Asp-376, and His-399. Consistent with a double-displacement mechanism common to alpha/beta-hydrolase fold enzymes, a covalent acetylenzyme intermediate was found. Together with secondary structure prediction highlighting an alpha/beta-hydrolase fold topology, our data provide strong evidence that OatC belongs to the alpha/beta-hydrolase fold family. This clearly distinguishes OatC from all other bacterial sialate O-acetyltransferases known so far because these are members of the hexapeptide repeat family, a class of acyltransferases that adopt a left-handed beta-helix fold and assemble into catalytic trimers.     

Eryptosis triggered by bismuth

Bismuth is used for multiple industrial purposes and in the treatment of several gastrointestinal diseases. Untoward effects of bismuth include anemia, which could, in theory, result from suicidal erythrocyte death or eryptosis. Hallmarks of eryptosis are cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Phosphatidylserine-exposing cells are rapidly cleared from circulating blood. Signaling leading to eryptosis includes increase in cytosolic Ca²⁺ activity and formation of ceramide. The present experiments explored whether bismuth elicits eryptosis. To this end, phosphatidylserine exposure was estimated from annexin V-binding, cell shrinkage from decrease of forward scatter in FACS analysis, cytosolic Ca²⁺ activity from Fluo3 fluorescence and ceramide abundance from binding of fluorescent antibodies. A 48 h exposure to bismuth (≥500 μg/l BiCl₃) enhanced the percentage of annexin V-binding cells and decreased forward scatter, increased cytosolic Ca²⁺ activity, and stimulated ceramide formation. In conclusion, bismuth stimulates eryptosis, the suicidal death of erythrocytes. The effect may contribute to or even account for the development of anemia during bismuth treatment. Moreover, ceramide formation in intestinal cells may participate in the therapeutic efficacy of bismuth preparations. This study was supported by the Deutsche Forschungsgemeinschaft SFB 766 and the Carl Zeiss Stiftung. The experimental work and studies on human subjects have been approved by the Ethical commission of the University of Tübingen.     

Autocatalytic Processing of m-AAA Protease Subunits in Mitochondria

m-AAA proteases are ATP-dependent proteolytic machines in the inner membrane of mitochondria which are crucial for the maintenance of mitochondrial activities. Conserved nuclear-encoded subunits, termed paraplegin, Afg3l1, and Afg3l2, form various isoenzymes differing in their subunit composition in mammalian mitochondria. Mutations in different m-AAA protease subunits are associated with distinct neuronal disorders in human. However, the biogenesis of m-AAA protease complexes or of individual subunits is only poorly understood. Here, we have examined the processing of nuclear-encoded m-AAA protease subunits upon import into mitochondria and demonstrate autocatalytic processing of Afg3l1 and Afg3l2. The mitochondrial processing peptidase MPP generates an intermediate form of Afg3l2 that is matured autocatalytically. Afg3l1 or Afg3l2 are also required for maturation of newly imported paraplegin subunits after their cleavage by MPP. Our results establish that mammalian m-AAA proteases can act as processing enzymes in vivo and reveal overlapping activities of Afg3l1 and Afg3l2. These findings might be of relevance for the pathogenesis of neurodegenerative disorders associated with mutations in different m-AAA protease subunits.