Enrichment and Physiological Characterization of a Novel Nitrospira-Like Bacterium Obtained from a Marine Sponge

Members of the nitrite-oxidizing genus Nitrospira are most likely responsible for the second step of nitrification, the conversion of nitrite (NO₂⁻) to nitrate (NO₃⁻), within various sponges. We succeeded in obtaining an enrichment culture of Nitrospira derived from the mesohyl of the marine sponge Aplysina aerophoba using a traditional cultivation approach. Electron microscopy gave first evidence of the shape and ultrastructure of this novel marine Nitrospira-like bacterium (culture Aa01). We characterized these bacteria physiologically with regard to optimal incubation conditions, especially the temperature and substrate range in comparison to other Nitrospira cultures. Best growth was obtained at temperatures between 28°C and 30°C in mineral medium with 70% North Sea water and a substrate concentration of 0.5 mM nitrite under microaerophilic conditions. The Nitrospira culture Aa01 is very sensitive against nitrite, because concentrations higher than 1.5 mM resulted in a complete inhibition of growth. Sequence analyses of the 16S rRNA gene revealed that the novel Nitrospira-like bacterium is separated from the sponge-specific subcluster and falls together with an environmental clone from Mediterranean sediments (98.6% similarity). The next taxonomically described species Nitrospira marina is only distantly related, with 94.6% sequence similarity, and therefore the culture Aa01 represents a novel species of nitrite-oxidizing bacteria.Numerous sponges have the capacity to accommodate large amounts of diverse microbes and represent significant sources for bioactive natural compounds (13). Many marine invertebrates excrete ammonium as a metabolic waste product (9), and the excretion of nitrite and nitrate has been taken as primary evidence that nitrifiers are active in these animals (10). By modulation of their pumping, sponges are a suitable habitat not only for aerobic microbes but also for anaerobic microbes. Accordingly, Hoffmann et al. (19) were able to detect major microbial pathways of the nitrogen cycle in the sponge Geodia barretti, including nitrification, the anammox process, and denitrification.Nitrification involves the biological oxidation of ammonia (NH₃) to nitrite (NO₂⁻) and further to nitrate (NO₃⁻) for energy purposes. It is of fundamental importance for the global nitrogen cycle in aquatic and terrestrial habitats. Nitrification is catalyzed by two phylogenetically distinct groups of microorganisms: in the first step, ammonia-oxidizing bacteria and archaea (AOB and AOA) take part in the oxidation of ammonia to nitrite, and in the second step nitrite-oxidizing bacteria (NOB) convert nitrite to nitrate (38).Nitrite has a central position in the nitrogen cycle, connecting aerobic and anaerobic pathways. Nitrite-oxidizing bacteria play a major role in removing nitrite from the environment because it is toxic for living organisms (31). Based on morphological characteristics, NOB have been divided into five genera. This classification also reflects the phylogenetic diversity of NOB, which includes Nitrobacter and Nitrococcus (Alpha- and Gammaproteobacteria), Nitrospina (putative Deltaproteobacteria), and the candidate genus “Candidatus Nitrotoga” (Betaproteobacteria) (2). The genus Nitrospira is more distantly related to the other known NOB because it is part of its own deep-branching bacterial phylum Nitrospirae. Marine species are present in all genera of NOB except in the newly identified genus “Candidatus Nitrotoga.”As all known nitrifying prokaryotes are slow growing and hard to maintain, their enrichment and isolation from environmental samples is difficult. Most physiological studies have been performed with pure cultures of a few “model” nitrifiers, in particular AOB related to the genus Nitrosomonas and NOB of the genus Nitrobacter. For the genus Nitrospira there are only four pure cultures available: the marine species Nitrospira marina (37), Nitrospira moscoviensis (12), “Candidatus Nitrospira bockiana” (25), and Nitrospira calida (E. Lebedeva, personal communication).Sponges of the family Aplysinidae contain large amounts of bacteria embedded within the sponge tissue matrix (15). For example, the biomass of Aplysina aerophoba consists of up to 40% bacteria (36). These sponges are able to differentiate between food bacteria and their own bacterial symbionts (41). Investigations of the diversity of sponge-associated bacteria, including different genetic and also cultivation approaches, have been made with several specimens (15, 16, 39). In terms of nitrification, Hentschel et al. (17) gave first evidence for the presence of nitrite oxidizers, and it has been verified that sponges harbor AOB and AOA (8). Most of the recognized NOB in sponges are Nitrospira-like bacteria (17, 32, 35), although in the beginning, there were further hints to 16S rRNA sequences, which are most closely related to Nitrospina gracilis (17). However, as these sequences were found only once, it could be assumed that Nitrospira is the main nitrite oxidizer in this environment. Nitrospira-like bacteria are deemed to be recalcitrant and fastidious, and they are easily overgrown by other bacteria under suboptimal conditions. Despite these limitations in the laboratory, Nitrospira was determined to be the most important nitrite oxidizer during wastewater treatment (21, 33), in aquaculture biofilters (14) and in freshwater systems (20, 29).Identification of sponge-associated microorganisms has been performed largely with culture-independent methods, which are 16S rRNA gene based (denaturing gradient gel electrophoresis [DGGE], terminal restriction fragment-length polymorphism [TRFLP]) or visual (fluorescence in situ hybridization [FISH], electron microscopy) (8, 11). Nevertheless, the cultivation of microorganisms is still essential for the investigation of their physiological potential and function in the environment. Information about physiological characteristics helps us to understand the metabolism and possible nutritional interactions of nitrifiers with the host sponge (8).This is the first report about cultivation of nitrifying bacteria originating from a marine sponge. We obtained a nitrite-oxidizing enrichment culture of a Nitrospira-like bacterium derived from Aplysina aerophoba, characterized it phylogenetically, and analyzed the most important physiological features.     

Synthesis of spirocyclic σ1 receptor ligands as potential PET radiotracers,structure–affinity relationships and in vitro metabolic stability

Several 3H-spiro[[2]benzofuran-1,4′-piperidines] bearing a p-fluorobenzyl residue at the N-atom and various substituents in position 3 of the benzofuran system were synthesized. The crucial reaction steps are the addition of a lithiated benzaldehyde derivative to the p-fluorobenzylpiperidone 5 and the BF₃·OEt₂ catalyzed substitution of the methoxy group of 2a by various nucleophiles. Structure–affinity relationship studies revealed that compounds with two protons (2d), a methoxy group (2a), and a cyano group (2e) in position 3 possess subnanomolar σ₁ affinity (K_i = 0.18 nM, 0.79 nM, 0.86 nM) and high selectivity against the σ₂ subtype. The metabolites of 2a, 2d, and 2e, which were formed upon incubation with rat liver microsomes, were identified. Additionally, the rate of metabolic degradation of 2a, 2d, and 2e was determined and compared with the degradation rate of the non-fluorinated spirocyclic compound 1. For the synthesis of the potential PET tracers [¹⁸F]2a and [¹⁸F]2e two different radiosynthetic approaches were followed.     

Osteopontin Is Cleaved at Multiple Sites Close to Its Integrin-binding Motifs in Milk and Is a Novel Substrate for Plasmin and Cathepsin D

Osteopontin (OPN) is a highly modified integrin-binding protein present in most tissues and body fluids where it has been implicated in numerous biological processes. A significant regulation of OPN function is mediated through phosphorylation and proteolytic processing. Proteolytic cleavage by thrombin and matrix metalloproteinases close to the integrin-binding Arg-Gly-Asp sequence modulates the function of OPN and its integrin binding properties. In this study, seven N-terminal OPN fragments originating from proteolytic cleavage have been characterized from human milk. Identification of the cleavage sites revealed that all fragments contained the Arg–Gly–Asp¹⁴⁵ sequence and were generated by cleavage of the Leu¹⁵¹–Arg¹⁵², Arg¹⁵²–Ser¹⁵³, Ser¹⁵³–Lys¹⁵⁴, Lys¹⁵⁴–Ser¹⁵⁵, Ser¹⁵⁵–Lys¹⁵⁶, Lys¹⁵⁶–Lys¹⁵⁷, or Phe¹⁵⁸–Arg¹⁵⁹ peptide bonds. Six cleavages cannot be ascribed to thrombin or matrix metalloproteinase activity, whereas the cleavage at Arg¹⁵²–Ser¹⁵³ matches thrombin specificity for OPN. The principal protease in milk, plasmin, hydrolyzed the same peptide bond as thrombin, but its main cleavage site was identified to be Lys¹⁵⁴–Ser¹⁵⁵. Another endogenous milk protease, cathepsin D, cleaved the Leu¹⁵¹–Arg¹⁵² bond. OPN fragments corresponding to plasmin activity were also identified in urine showing that plasmin cleavage of OPN is not restricted to milk. Plasmin, but not cathepsin D, cleavage of OPN increased cell adhesion mediated by the α_Vβ₃- or α₅β₁-integrins. Similar cellular adhesion was mediated by plasmin and thrombin-cleaved OPN showing that plasmin can be a potent regulator of OPN activity. These data show that OPN is highly susceptible to cleavage near its integrin-binding motifs, and the protein is a novel substrate for plasmin and cathepsin D.     

Extracellular superoxide dismutase

The extracellular space is protected from oxidant stress by the antioxidant enzyme extracellular superoxide dismutase (EC-SOD), which is highly expressed in selected tissues including blood vessels, heart, lungs, kidney and placenta. EC-SOD contains a unique heparin-binding domain at its carboxy-terminus that establishes localization to the extracellular matrix where the enzyme scavenges superoxide anion. The EC-SOD heparin-binding domain can be removed by proteolytic cleavage, releasing active enzyme into the extracellular fluid. In addition to protecting against extracellular oxidative damage, EC-SOD, by scavenging superoxide, preserves nitric oxide bioactivity and facilitates hypoxia-induced gene expression. Loss of EC-SOD activity contributes to the pathogenesis of a number of diseases involving tissues with high levels of constitutive extracellular superoxide dismutase expression. A thorough understanding of the biological role of EC-SOD will be invaluable for developing novel therapies to prevent stress by extracellular oxidants.     

Production of a lipolytic enzyme originating from Bacillus halodurans LBB2 in the methylotrophic yeast Pichia pastoris

A gene encoding a lipolytic enzyme amplified from the alkaliphilic bacterium Bacillus halodurans LBB2 was cloned into the pPICZαB vector and integrated into the genome of the protease deficient yeast strain Pichia pastoris SMD1168H. This previously undescribed enzyme was produced in active form, and cloning in frame with the Saccharomyces cerevisiae secretion signal (α-factor) enabled extracellular accumulation of correctly processed enzyme, with an apparent molecular mass of 30 kDa. In shake-flask cultivations, very low production levels were obtained, but these were significantly improved by use of a “batch-induced” cultivation technique which allowed a maximum enzyme activity of 14,000 U/l using p-nitrophenyl butyrate (C-4) as a substrate and a final extracellular lipolytic enzyme concentration of approximately 0.2 g/l. Partial characterization of the produced enzyme (at pH 9) revealed a preference for the short-chain ester (C-4) and significant but lower activity towards medium (C5-C6) and long (C16 and C18) fatty acid chain-length esters. In addition, the enzyme exhibited true lipase activity (7,300 U/l) using olive oil as substrate and significant levels of phospholipase activity (6,400 U/l) by use of a phosphatidylcholine substrate, but no lysophospholipase activity was detected using a lysophosphatidylcholine substrate.     

Expression pattern of Filamin-240 in Drosophila blood cells

The expression pattern of Filamin-240 was studied in subsets of Drosophila blood cells by means of immunofluorescent staining and Western blot analysis with use of an antibody specific to a "filamin-folding domain", a consensus motif profile generated from the 20 existing filamin repeats. Expression of Filamin-240 is restricted to lamellocytes - a special blood cell type of the cellular immune response - and is involved in the regulation of lamellocyte development. In the cher1 homozygous larvae, which lack Filamin-240 protein, a vigorous lamellocyte differentiation occurs which is further enhanced upon in vivo immune challenge by a parasitic wasp, Leptopilina boulardi. By introducing a full-length transgene encoding the Drosophila Filamin-240 protein into the cher1 Filamin-deficient homozygous mutant, the mutant blood cell phenotype was rescued. These data demonstrate that the expression of Filamin-240 is strictly lamellocyte specific in Drosophila blood cells and that the protein is a suppressor of lamellocyte development.     

TGF-β1 suppresses IL-6-induced STAT3 activation through regulation of Jak2 expression in prostate epithelial cells

Chronic inflammation plays an important role in the initiation and progression of various human diseases including benign prostatic hyperplasia or prostate cancer. Here we show that the proinflammatory cytokine interleukin-6 (IL-6) has prosurvival effects and chronically activates the Jak2/STAT3 signalling pathway in a model of benign prostatic hyperplasia (BPH-1). We demonstrate that the antiinflammatory cytokine transforming growth factor-β1 (TGF-β1), which also permanently activates its canonical signalling pathway through SMAD proteins in BPH-1 cells, modifies the effects of IL-6 on cell proliferation. Importantly, TGF-β1 inhibits IL-6 signal transduction by decreasing the phosphorylation levels of STAT3. This effect is associated with decreased expression of Jak2 at both mRNA and protein levels. Moreover, we showed that TGF-β1 inhibits IL-6-induced expression of the cancer-associated gene MUC1. These observations demonstrated a novel interaction between TGF-β1 and IL-6 signalling and suggested another mechanism of how defects in TGF-β signalling, frequently associated with prostate pathologies, can contribute to the disruption of tissue homeostasis.