[Health promotion aspects in reorganization of a centralized operating department]. / Gesundheitsfördernde Aspekte bei der Reorganisation einer zentralen Operationsabteilung.

In many health care institutions (eg, hospitals), employees' health and satisfaction are not the main concern. However, if health and productivity are affected by high sickness rates and low motivation, health and satisfaction of the employees become important. This applies especially to hospitals engaged in acute patient care. We showed that a complete reorganisation of the central O. R. resulted in employees' working satisfaction, low illness rates (- 70 %) and increased motivation. In addition, the new structure increased the efficacy (> 500 operations more per year), improved the economy (savings of 500,000 annually) and increased patient satisfaction as expected and desired.

1.68-A crystal structure of endopolygalacturonase II from Aspergillus niger and identification of active site residues by site-directed mutagenesis.

Polygalacturonases specifically hydrolyze polygalacturonate, a major constituent of plant cell wall pectin. To understand the catalytic mechanism and substrate and product specificity of these enzymes, we have solved the x-ray structure of endopolygalacturonase II of Aspergillus niger and we have carried out site-directed mutagenesis studies. The enzyme folds into a right-handed parallel beta-helix with 10 complete turns. The beta-helix is composed of four parallel beta-sheets, and has one very small alpha-helix near the N terminus, which shields the enzyme's hydrophobic core. Loop regions form a cleft on the exterior of the beta-helix. Site-directed mutagenesis of Asp(180), Asp(201), Asp(202), His(223), Arg(256), and Lys(258), which are located in this cleft, results in a severe reduction of activity, demonstrating that these residues are important for substrate binding and/or catalysis. The juxtaposition of the catalytic residues differs from that normally encountered in inverting glycosyl hydrolases. A comparison of the endopolygalacturonase II active site with that of the P22 tailspike rhamnosidase suggests that Asp(180) and Asp(202) activate the attacking nucleophilic water molecule, while Asp(201) protonates the glycosidic oxygen of the scissile bond.

Crystal structure of a murine alpha-class glutathione S-transferase involved in cellular defense against oxidative stress.

Glutathione S-transferases (GSTs) are ubiquitous multifunctional enzymes which play a key role in cellular detoxification. The enzymes protect the cells against toxicants by conjugating them to glutathione. Recently, a novel subgroup of alpha-class GSTs has been identified with altered substrate specificity which is particularly important for cellular defense against oxidative stress. Here, we report the crystal structure of murine GSTA4-4, which is the first structure of a prototypical member of this subgroup. The structure was solved by molecular replacement and refined to 2.9 A resolution. It resembles the structure of other members of the GST superfamily, but reveals a distinct substrate binding site.

Crystallization and preliminary crystallographic characterization of endo-polygalacturonase II from Aspergillus niger.

The endo-polygalacturonase II from Aspergillus niger has been crystallized from an ammonium sulfate solution by the hanging drop method. The crystals belong to the monoclinic space group P2(1), with cell dimensions a = 69.6 A, b = 152.6 A, c = 74.0 A and beta = 91.2 degrees with four molecules per asymmetric unit. The crystals diffract to at least 2.8 A resolution and are suitable for X-ray analysis.

Characterization of a Shaw-related potassium channel family in rat brain.

Previously, we characterized a Shaker-related family of voltage-gated potassium channels (RCK) in rat brain. Now, we describe a second family of voltage-gated potassium channels in the rat nervous system. This family is related to the Drosophila Shaw gene and has been dubbed Raw. In contrast to the RCK potassium channel family the Raw family utilizes extensive alternative splicing for expressing potassium channel subunits with variant C-termini. These alternative C-termini do not appear to influence the electrophysiological and pharmacological properties as studied in the Xenopus oocyte expression system. In situ hybridizations to sections of rat brain indicate that members of the Raw family are expressed in distinct areas of the central nervous system. Probably, Raw channels are expressed predominantly as homomultimers. Immunocytochemical experiments with antibodies against Raw3 and RCK4 proteins which form two distinct A-type potassium channels indicate that in hippocampus the two channels are expressed both in different neurons and in the same ones. In general, properties of Raw potassium channels appeared to be similar to RCK channels. However, Raw outward currents, in contrast to RCK currents, exhibit an intense rectification at test potentials higher than +20 to +40 mV. RCK and Raw channel subunits did not measurably coassemble into RCK/Raw heteromultimers after coinjecting RCK and Raw cRNA into Xenopus oocytes. These results suggest that members of the RCK and the Raw potassium channel families express potassium channels which form independent outward current systems. Combining the results of in situ hybridizations, immunocytochemical staining and expression of the cloned potassium channels in Xenopus oocytes demonstrates that unrestrained mixing of potassium channel subunits to form hybrid channels does not occur in the rat central nervous system. A single neuron is able to express multiple, independently assembled potassium channels.

Swapping of functional domains in voltage-gated K+ channels.

Functionally significant properties of domains in the amino acid sequence of potassium (K+) channel-forming proteins have been investigated by constructing chimeric K+ channels. The N-terminal domain of ShA2 channels was responsible for the fast inactivation (IKA) and also determined a shift in the threshold of activation whereas the membrane domain determined the timecourse of slow inactivation. The binding site for dendrotoxin (DTX), but not for mast cell degranulating peptide (MCDP), is completely located on the loop between the membrane spanning segments S5 and S6 in RCK1 channels. A certain part of this region which has recently been designated as a narrow part of the pore was found to be not responsible for the differences in the single-channel current amplitude between RCK4 and RCK2 K+ channels. Interchange of the C-terminal domain did not influence activation or inactivation of the channels.

Cloning and functional expression of a TEA-sensitive A-type potassium channel from rat brain.

A rat brain cDNA (Raw3) related to the Drosophila Shaw K+ channel family has been characterized. Raw3 cRNA leads to the formation of TEA-insensitive, fast inactivating (A-type) K+ channels when injected into Xenopus laevis oocytes. Raw3 channels have markedly different properties from the previously cloned rat A-type K+ channel RCK4, Raw3 channels operate in the positive voltage range.

Heteromultimeric channels formed by rat brain potassium-channel proteins.

An important step towards understanding the molecular basis of the functional diversity of voltage-gated K+ channels in the mammalian brain has been the discovery of a family of genes encoding rat brain K+ channel-forming (RCK) proteins. All species of these RCK proteins form homomultimeric voltage-gated K+ channels with distinct functional characteristics in Xenopus laevis oocytes following injection of the respective cRNAs. RCK-specific mRNAs are coexpressed in several regions of the brain, suggesting that RCK proteins also assemble into heteromultimeric K+ channels. In addition expression experiments with fractionated poly(A)+ mRNA have suggested that heteromultimeric K+ channels may occur in mammalian brain. We report here that heteromultimeric K+ channels composed of two different RCK proteins (RCK1 and RCK4) assemble after cotransfection of HeLa cells with the corresponding cDNAs and after coinjection of the corresponding cRNAs into Xenopus oocytes. The heteromultimeric RCK1, 4 channel mediates a transient potassium outward current, similar to the RCK4 channel but inactivates more slowly, has a larger conductance and is more sensitive to block by dendrotoxin and tetraethylammonium chloride.

Cloning and expression of a human voltage-gated potassium channel. A novel member of the RCK potassium channel family.

We have isolated and characterized a human cDNA (HBK2) that is homologous to novel member (RCK2) of the K+ channel RCK gene family expressed in rat brain. RCK2 mRNA was detected predominantly in midbrain areas and brainstem. The primary sequences of the HBK2/RCK2 K+ channel proteins exhibit major differences to other members of the RCK gene family. The bend region between segments S1 and S2 is unusually long and does not contain the N-glycosylation site commonly found in this region. They might be O-glycosylated instead. Functional characterization of the HBK2/RCK2 K+ channels in Xenopus laevis oocytes following micro-injection in in vitro transcribed HBK2 or RCK2 cRNA showed that the HBK2/RCK2 proteins form voltage-gated K+ channels with novel functional and pharmacological properties. These channels are different to RCK1, RCK3, RCK4 and RCK5 K+ channels.

Molecular basis of functional diversity of voltage-gated potassium channels in mammalian brain.

Cloning and sequencing of cDNAs isolated from a rat cortex cDNA library reveals that a gene family encodes several highly homologous K+ channel forming (RCK) proteins. Functional characterization of the channels expressed in Xenopus laevis oocytes following microinjection of in vitro transcribed RCK-specific RNAs shows that each of the RCK proteins forms K+ channels that differ greatly in both their functional and pharmacological properties. This suggests that the molecular basis for the diversity of voltage-gated K+ channels in mammalian brain is based, at least partly, on the expression of several RCK proteins by a family of genes and their assembly to homooligomeric K+ channels with different functional properties.