[Nontraumatic resuscitation room management of critically ill patients]. / Nichttraumatologisches Schockraummanagement.

Critically ill patients are often initially treated by out-of-hospital emergency medicine services. A major challenge-especially at the interface between out-of-hospital and in-hospital care-is to continue patient care without wasting time, while maintaining a high level. These include the stabilization of vital functions (e.g., airway management, noninvasive/invasive ventilation, circulatory stabilization) and implementation of a suitable diagnostic and therapeutic strategy (e.g., laboratory examinations, sonography, radiological imaging). In recent years, therefore, interest and research has focused on the topic of "nontraumatic resuscitation room care". The first monocentric data recently became available and work is ongoing to develop nontraumatic resuscitation room management for optimal care of critically ill patients in the emergency department. Based on initial studies, experiences and expert opinions, this paper describes a structured approach to nontraumatic resuscitation room management.

Specificities of functionally expressed chalcone and acridone synthases from Ruta graveolens.

The common rue, Ruta graveolens L., expresses two types of closely related polyketide synthases that condense three malonyl-CoAs with N-methylanthraniloyl-CoA or 4-coumaroyl-CoA to produce acridone alkaloids and flavonoid pigments, respectively. Two acridone synthase cDNAs (ACS1 and ACS2) have been cloned from Ruta cell cultures, and we report now the cloning of three chalcone synthase cDNAs (CHS1 to CHS3) from immature Ruta flowers. The coding regions of these three cDNAs differ only marginally, and the translated polypeptides show about 90% identity with the CHSs from Citrus sinensis but less than 75% with the Ruta endogeneous ACSs. CHS1 was functionally expressed in Eschericha coli and its substrate specificity compared with those of the recombinant ACS1 and ACS2. 4-Coumaroyl-CoA was the preferred starter substrate for CHS1, but cinnamoyl-CoA and caffeoyl-CoA were also turned over at significant rates. However, N-methylanthraniloyl-CoA was not accepted. In contrast, highly active preparations of recombinant ACS1 or ACS2 showed low, albeit significant, CHS side activities with 4-coumaroyl-CoA, which on average reached 16% (ACS1) and 12% (ACS2) of the maximal activity determined with N-methylanthraniloyl-CoA as the starter substrate, while the conversion of cinnamoyl-CoA was negligible with both ACSs. The condensation mechanism of the acridone ring system differs from that of chalcone/flavanone formation. Nevertheless, our results suggest that very minor changes in the sequences of Ruta CHS genes are sufficient to also accommodate the formation of acridone alkaloids, which will be investigated further by site-directed mutagenesis.

Purification of recombinant flavanone 3beta-hydroxylase from petunia hybrida and assignment of the primary site of proteolytic degradation.

Flavanone 3beta-hydroxylase catalyzes the Fe(II)/oxoglutarate-dependent hydroxylation of (2S)-flavanones to (2R,3R)-dihydroflavonols in the course of flavonol/anthocyanin or catechin biosynthesis. The enzyme from Petunia hybrida consists of a 41,655-Da polypeptide that is prone to rapid proteolysis in crude plant extracts as well as on expression in Escherichia coli, and commercial protease inhibitors were inefficient in stopping the degradation. To pinpoint the primary site of proteolysis and to improve the activity yields, two revised schemes of purification were developed for the recombinant polypeptides. Applying a four-step protocol based on extraction and ion-exchange chromatography at pH 7.5, the primary, catalytically inactive proteolytic enzyme fragment (1.1 mg) was isolated and shown to cross-react on Western blotting as one homogeneous band of about 38 kDa. Mass spectrometric analysis assigned a mass of 37,820 +/- 100 Da to this fragment, and partial sequencing revealed an unblocked amino terminus identical to that of the native 3beta-hydroxylase. Thus, the native enzyme had been degraded by proteolysis of a small carboxy-terminal portion, and the primary site of cleavage must be assigned most likely to the Glu 337-Leu 338 bond, accounting for a loss of about 3800 Da. Alternatively, the enzyme degradation was greatly reduced when the extraction of recombinant bacteria was carried out with phosphate buffer at pH 5.5 followed by size exlusion and anion-exchange chromatography. This rapid, two-step purification resulted in a homogeneous 3beta-hydroxylase of high specific acitivity (about 32 mkat/kg) at roughly 5% yield, and the procedure is a major breakthrough in mechanistic investigations of this class of labile dioxygenases.

The monomeric polypeptide comprises the functional flavanone 3beta-hydroxylase from Petunia hybrida.

Flavanone 3beta-hydroxylase catalyzes the Fe(II)/oxoglutarate-dependent hydroxylation of (2S)-flavanones to (2R,3R)-dihydroflavonols in the biosynthesis of flavonoids, catechins and anthocyanidins. The enzyme had been partially purified from Petunia hybrida and proposed to be active as a dimer of roughly 75 kDa in size. More recently, the Petunia 3beta-hydroxylase was cloned and shown to be encoded in a 41655 Da polypeptide. In order to characterize the molecular composition, the enzyme was expressed in a highly active state in Escherichia coli and purified to apparent homogeneity. Size exclusion chromatographies of the pure, recombinant enzyme revealed that this flavanone 3beta-hydroxylase exists in functional monomeric and oligomeric forms. Protein cross-linking experiments employing a specific homobifunctional sulfhydryl group reagent or the photochemical activation of tryptophan residues confirmed the tendency of the enzyme to aggregate to oligomeric complexes in solution. Thorough equilibrium sedimentation analyses, however, revealed a molecular mass of 39. 2+/-12 kDa for the recombinant flavanone 3beta-hydroxylase. The result implies that the monomeric polypeptide comprises the catalytically active flavanone 3beta-hydroxylase of P. hybrida, which may readily associate in vivo with other proteins.

Site-directed mutagenesis of the active site serine290 in flavanone 3beta-hydroxylase from Petunia hybrida.

Flavanone 3beta-hydroxylase (FHT) catalyzes a pivotal reaction in the formation of flavonoids, catechins, proanthocyanidins and anthocyanidins. In the presence of oxygen and ferrous ions the enzyme couples the oxidative decarboxylation of 2-oxoglutarate, releasing carbon dioxide and succinate, with the oxidation of flavanones to produce dihydroflavonols. The hydroxylase had been cloned from Petunia hybrida and expressed in Escherichia coli, and a rapid isolation method for the highly active, recombinant enzyme had been developed. Sequence alignments of the Petunia hydroxylase with various hydroxylating 2-oxoglutarate-dependent dioxygenases revealed few conserved amino acids, including a strictly conserved serine residue (Ser290). This serine was mutated to threonine, alanine or valine, which represent amino acids found at the corresponding sequence position in other 2-oxoglutarate-dependent enzymes. The mutant enzymes were expressed in E. coli and purified to homogeneity. The catalytic activities of [Thr290]FHT and [Ala290]FHT were still significant, albeit greatly reduced to 20 and 8%, respectively, in comparison to the wild-type enzyme, whereas the activity of [Val290]FHT was negligible (about 1%). Kinetic analyses of purified wild-type and mutant enzymes revealed the functional significance of Ser290 for 2-oxoglutarate-binding. The spatial configurations of the related Fe(II)-dependent isopenicillin N and deacetoxycephalosporin C synthases have been reported recently and provide the lead structures for the conformation of other dioxygenases. Circular dichroism spectroscopy was employed to compare the conformation of pure flavanone 3beta-hydroxylase with that of isopenicillin N synthase. A double minimum in the far ultraviolet region at 222 nm and 208-210 nm and a maximum at 191-193 nm which are characteristic for alpha-helical regions were observed, and the spectra of the two dioxygenases fully matched revealing their close structural relationship. Furthermore, the spectrum remained unchanged after addition of either ferrous ions, 2-oxoglutarate or both of these cofactors, ruling out a significant conformational change of the enzyme on cofactor-binding.