[Evading the catastrophy]. / An der Katastrophe vorbei.

A 21-year-old athletic woman had been suffering from flank pain during fluid intake after sports for some time. Urological work-up revealed hydronephrosis with a "fishhook" shape and medial displacement of the ureter. This ureteral narrowing was studied ureterorenoscopically, bioptically and endoscopic-radiologically without the correct diagnosis of a retrocaval ureter being made. Therapeutically, a DJ stent was inserted several times, a balloon dilatation was performed under anaesthesia three times and finally a permanent DJ catheter was inserted. Due to the patient's dissatisfaction, an endoscopic endopyelotomy using Acucise was offered.

[Life-threatening complication caused by a neurostimulator]. / Lebensbedrohliche Komplikation durch Neurostimulator.

We report on a 66-year-old neuro-urological female patient who, three years after implantation of a neurostimulator, experienced cecal necrosis due to strangulation caused by the cable of the device.

Metabolic deficiences revealed in the biotechnologically important model bacterium Escherichia coli BL21(DE3).

The Escherichia coli B strain BL21(DE3) has had a profound impact on biotechnology through its use in the production of recombinant proteins. Little is understood, however, regarding the physiology of this important E. coli strain. We show here that BL21(DE3) totally lacks activity of the four [NiFe]-hydrogenases, the three molybdenum- and selenium-containing formate dehydrogenases and molybdenum-dependent nitrate reductase. Nevertheless, all of the structural genes necessary for the synthesis of the respective anaerobic metalloenzymes are present in the genome. However, the genes encoding the high-affinity molybdate transport system and the molybdenum-responsive transcriptional regulator ModE are absent from the genome. Moreover, BL21(DE3) has a nonsense mutation in the gene encoding the global oxygen-responsive transcriptional regulator FNR. The activities of the two hydrogen-oxidizing hydrogenases, therefore, could be restored to BL21(DE3) by supplementing the growth medium with high concentrations of Ni²âº (Ni²âº-transport is FNR-dependent) or by introducing a wild-type copy of the fnr gene. Only combined addition of plasmid-encoded fnr and high concentrations of MoO4²â» ions could restore hydrogen production to BL21(DE3); however, to only 25-30% of a K-12 wildtype. We could show that limited hydrogen production from the enzyme complex responsible for formate-dependent hydrogen evolution was due solely to reduced activity of the formate dehydrogenase (FDH-H), not the hydrogenase component. The activity of the FNR-dependent formate dehydrogenase, FDH-N, could not be restored, even when the fnr gene and MoO4²â» were supplied; however, nitrate reductase activity could be recovered by combined addition of MoO4²â» and the fnr gene. This suggested that a further component specific for biosynthesis or activity of formate dehydrogenases H and N was missing. Re-introduction of the gene encoding ModE could only partially restore the activities of both enzymes. Taken together these results demonstrate that BL21(DE3) has major defects in anaerobic metabolism, metal ion transport and metalloprotein biosynthesis.

Efficient electron transfer from hydrogen to benzyl viologen by the [NiFe]-hydrogenases of Escherichia coli is dependent on the coexpression of the iron-sulfur cluster-containing small subunit.

Escherichia coli can both oxidize hydrogen and reduce protons. These activities involve three distinct [NiFe]-hydrogenases, termed Hyd-1, Hyd-2, and Hyd-3, each minimally comprising heterodimers of a large subunit, containing the [NiFe] active site, and a small subunit, bearing iron-sulfur clusters. Dihydrogen-oxidizing activity can be determined using redox dyes like benzyl viologen (BV); however, it is unclear whether electron transfer to BV occurs directly at the active site, or via an iron-sulfur center in the small subunit. Plasmids encoding Strep-tagged derivatives of the large subunits of the three E. coli [NiFe]-hydrogenases restored activity of the respective hydrogenase to strain FTD147, which carries in-frame deletions in the hyaB, hybC, and hycE genes encoding the large subunits of Hyd-1, Hyd-2, and Hyd-3, respectively. Purified Strep-HyaB was associated with the Hyd-1 small subunit (HyaA), and purified Strep-HybC was associated with the Hyd-2 small subunit (HybO), and a second iron-sulfur protein, HybA. However, Strep-HybC isolated from a hybO mutant had no other associated subunits and lacked BV-dependent hydrogenase activity. Mutants deleted separately for hyaA, hybO, or hycG (Hyd-3 small subunit) lacked BV-linked hydrogenase activity, despite the Hyd-1 and Hyd-2 large subunits being processed. These findings demonstrate that hydrogenase-dependent reduction of BV requires the small subunit.

Development of a cell-free system reveals an oxygen-labile step in the maturation of [NiFe]-hydrogenase 2 of Escherichia coli.

By combining extracts from strains lacking genes encoding either the maturation enzymes or the large subunits of hydrogenases 1, 2 and 3 we could reconstitute in vitro under strictly anaerobic conditions 10-15% of the hydrogenase activity present in wild type Escherichia coli extracts. Purified, unprocessed Strep-tagged variants of the hydrogenase 2 large subunit, HybC, isolated from either a ΔhybD (encoding the hydrogenase 2-specific protease) mutant or a strain deficient in HypF could also be matured to active, processed enzyme using this system. These studies reveal that minimally one step early on the hydrogenase maturation pathway is oxygen-labile.