[Water contamination and infection rate after water births]. / Die Kontamination des Wassers und die Infektionsrate bei der Wassergeburt.

OBJECTIVE: This study aimed at examining the water of the birthing tub for pathogenic germs and at comparing the infection rates of the children born conventionally. METHOD: In a prospective study, the germs found in the water of 300 water births were determined . The tub water was sampled twice: sample A was taken after filling the tub with tap water, sample B after the water delivery. In addition, the paediatrician documented any signs of infection of the neonates during their hospital stay. RESULTS: The A samples contained Legionella in 29%, Pseudomonas aeruginosa in 22%, enterococci in 18%, colibacilli in 32% and Escherichia coli in 8%. After fitting a filter system, no Legionella was detected any more. P. aeruginosa was found in only 3% of the samples. In the B samples, we found an increased contamination by colibacilli in 81%. A marked burden with E. coli was detected in 58% of the samples. Due to a clinically an biochemically suspected beginning infection, 1.15% of the water-born children (14 out of 1,215) were given antibiotics. In contrast, 2.30% of the conventionally born neonates (19 out of 817) were treated with antibiotics. CONCLUSION: It is evident that during the bearing-down phase faeces are discharged into the birthing tube and that the water is contaminated mainly by E. coli and colibacilli, but also slightly by Staphylococcus aureus. The contamination of the tap water by Legionella and Pseudomonas could clearly be reduced by installation of a filter system into the supply hose of the birthing tub. Neonatal infections were not more frequent after water births than after conventional deliveries.

Molecular epidemiology of Legionella pneumophila environmental isolates representing nine different serogroups determined by automated ribotyping and pulsed-field gel electrophoresis.

The purposes of the study were (i) to describe the abundance and epidemiology of Legionellaceae in the man-made environment in a northern Italian area, (ii) to assess the concordance between pulsed-field gel electrophoresis (PFGE) and automated ribotyping (AR) techniques for genotyping L. pneumophila and (iii) to investigate the correlation between serogrouping and genotyping data. Water was sampled from reservoirs in 12 buildings across an area of 80-km radius. Despite the water temperature always being maintained above 55 degrees C, all of the buildings sampled were contaminated with Legionellaceae on at least one occasion and 63 L. pneumophila isolates representing nine different serogroups were collected. The two DNA methods revealed a high degree of genetic heterogeneity, even though identical L. pneumophila clones were recovered at different sites. The AR technique provided a fairly reliable approximation of PFGE results (73% concordance), however there was poor correlation between serogrouping and genotyping data as identical DNA fingerprints were shared by isolates of different serogroups.

Review of 1600 water births. Does water birth increase the risk of neonatal infection?

OBJECTIVES: We reviewed 1600 water births at a single institution over an 8-year period. METHODS: We compared 737 primiparae deliveries in water with 407 primiparae deliveries in bed, and 142 primiparae on the delivery stool. We also evaluated the duration of labor, perineal trauma, arterial cord blood pH, postpartum maternal hemoglobin levels, and rates of neonatal infection. In 250 water deliveries we performed bacterial cultures of water samples obtained from the bath after filling and after delivery. RESULTS: The duration of the first stage of labor was significantly shorter with a water birth than with a land delivery (380 vs. 468 minutes, P<0.01). The episiotomy rate in all water births was lower with a water birth than with a delivery in bed or a delivery on the birthing stool (0.38%, 23%, and 8.4%, respectively). The rate of perineal tears was similar (23%, respectively). There were no differences in the duration of the second stage (34 vs. 37 minutes), arterial cord blood pH, or postpartum maternal hemoglobin levels. No woman using the water birth method required analgesics. The rate of neonatal infection was also not increased with a water birth (1.22% vs. 2.64%, respectively). CONCLUSION: Water birth appears to be associated with a significantly shorter first stage of labor, lower episiotomy rate and reduced analgesic requirements when compared with other delivery positions. If women are selected appropriately and hygiene rules are respected, water birth appears to be safe for both the mother and neonate.

[Water birth and neonatal infections. Experience with 1575 deliveries in water]. / Partorire in acqua e rischio di infezione. Esperienza dopo 1575 parti in acqua.

AIM: The aim of our study is to provide an answer on the advantages offered by water births, to compare them with 2 other delivery positions and to analyse the pathogenous microorganisms present in the water from the bath. METHODS: We compared 725 primiparae deliveries in water, 407 primiparae deliveries in bed and 142 on the delivery stool over the last 7 years. We evaluated the duration of labour, perineal trauma, arterial cord blood pH, shoulder dystocia and postpartum maternal hemoglobin levels. We have evaluated 200 water samples, taken from the bath after filling it and after delivery, and analyzed the pathogenous microorganisms and the possibility of neonatal infections. RESULTS: The duration (first stage) of labour and the rate of episiotomies was significantly reduced in primiparae delivering in water compared with the other delivery positions. Nevertheless, the percentage of perineal trauma was not increased. There were no differences in the duration of the second stage and arterial umbilical cord blood pH. Postpartum maternal hemoglobin levels remained unchanged. No woman delivering in the water required analgesics. Infections after water births do not occur more frequently than after traditional births. CONCLUSIONS: Our results show that water birth has major advantages compared with traditional delivery methods. It is associated with a significantly shorter first stage of labour, a lower episiotomy rate and reduced analgesic requirements when compared with other delivery positions. Provided that the women are selected appropriately, and the hygiene rules are respected, water birth is safe for mother and neonate.

Azobenzene as photoresponsive conformational switch in cyclic peptides.

Over the last decades azobenzene has been the most widely used optical trigger for the synthesis of photoresponsive systems ranging from poly-alpha-amino acids to innovative materials with light-controlled mechanical and optical properties. More recently, its use in form of appropriate derivatives allowed to generate cyclic peptide structures of constraint conformational space and thus to exploit its reversible photoisomerization to induce well defined transitions between different conformational states. These can be characterized in detail in both photostationary states making such systems ideal substrates for ultrafast spectroscopic analysis of conformational transitions. Moreover, the changes in biophysical properties that occur as a consequence of the different conformational states can be exploited for a photo-control of a large variety of molecular recognition processes.

Ultrafast conformational dynamics in cyclic azobenzene peptides of increased flexibility.

Structural changes of peptides containing the azobenzene dye 4-aminomethyl-phenylazobenzoic acid (AMPB) are studied with ultrafast spectroscopy. AMPB peptides are a new class of molecules where the photoisomerizable dye azobenzene is linked to the peptide moiety via a flexible methylene spacer. The ultrafast reactions in the femtosecond to nanosecond time domain are investigated for the optical switch AMPB, a linear and cyclic octapeptide, and a bicyclic octapeptide containing an additional disulfide bridge. These molecules with increasing conformational constraints are studied for the cis to trans and the trans to cis photoreactions. For the cis to trans reaction the isomerization of the chromophore occurs fast in the 1-ps range, whereas it is slower (10-ps range) in the trans to cis reaction. In all peptides the structural changes of the chromophore lead to modifications in the peptide structure in the 10-ps-1-ns time range. The results indicate that the chromophore AMPB acts simultaneously as a fast molecular switch and as a sensor for initial conformational dynamics in the peptide. Experiments in the mid-infrared range where the structural changes of the peptide backbone are directly observed demonstrate that the essential part of the structural dynamics in the bicyclic AMPB peptide occurs faster than 10 ns.

Unusual properties of the fungal conventional kinesin neck domain from Neurospora crassa.

Fungal conventional kinesins are unusually fast microtubule motor proteins. To compare the functional organization of fungal and animal conventional kinesins, a set of C-terminal deletion mutants of the Neurospora crassa conventional kinesin, NcKin, was investigated for its biochemical and biophysical properties. While the shortest, monomeric construct comprising the catalytic core and the neck-linker (NcKin343) displays very high steady-state ATPase (k(cat) = 260/s), constructs including both the full neck and adjacent hinge domains (NcKin400, NcKin433 and NcKin480) show wild-type behaviour: they are dimeric, show fast gliding and slower ATP turnover rates (k(cat) = 60-84/s), and are chemically processive. Unexpectedly, a construct (NcKin378, corresponding to Drosophila KHC381) that includes just the entire coiled-coil neck is a monomer. Its ATPase activity is slow (k(cat) = 27/s), and chemical processivity is abolished. Together with a structural analysis of synthetic neck peptides, our data demonstrate that the NcKin neck domain behaves differently from that of animal conventional kinesins and may be tuned to drive fast, processive motility.

N,N'-di-tert-butoxycarbonyl-1H-benzotriazole-1-carboxamidine derivatives are highly reactive guanidinylating reagents.

By enhancing the leaving group character of benzotriazole via electron-withdrawing substituents such as the 5-chloro or 6-nitro derivatives with the related N,N'-di-tert-butoxycarbonyl-1H-benzotriazole-1-carboxamidines, highly efficient reagents are obtained for conversion of primary and secondary amines in solution and in solid phase to diprotected guanidines. [reaction: see text]

Analytical solution to the Lipari-Szabo model based on the reduced spectral density approximation offers a novel protocol for extracting motional parameters.

An analytical solution to the Lipari-Szabo model is derived for isotropic overall tumbling. The parameters of the original Lipari-Szabo model, the order parameter S2 and the effective internal correlation time tau(e), are calculated from two values of the spectral density function. If additionally the spectral density value J(0) is known, the exchange contribution R(ex) term can also be determined. The overall tumbling time tau(c) must be determined in advance, for example, from T1/T2 ratios. The required spectral density values are obtained by reduced spectral density mapping from T1, T2, and NOE measurements. Our computer simulations show that the reduced spectral density mapping is a very good approximation in almost all cases in which the Lipari-Szabo model is applicable. The robustness of the analytical formula to experimental errors is also investigated by extensive computer simulations and is found to be similar to that of the fitting procedures. The derived formulas were applied to the experimental 15N relaxation data of ubiquitin. Our results agree well with the published parameter values of S2 and tau(e), which were obtained from standard fitting procedures. The analytical approach to extract parameters of molecular motions may be more robust than standard analyses and provides a safeguard against spurious fitting results, especially for determining the exchange contribution R(ex).

Sarcolipin, the shorter homologue of phospholamban, forms oligomeric structures in detergent micelles and in liposomes.

The human 31-amino acid integral membrane protein sarcolipin (SLN), which regulates the sarcoplasmic reticulum Ca-ATPase in fast-twitch skeletal muscle, was chemically synthesized. Appropriate synthesis and purification strategies were used to achieve high purity and satisfactory yields of this hydrophobic and poorly soluble protein. Structural and functional properties of SLN were analyzed and compared with the homologous region of human phospholamban (PLB) comprising residues Ala(24)-Leu(52) (PLB-(24-52)), the regulatory protein of the cardiac sarcoplasmic reticulum Ca-ATPase. Circular dichroism spectroscopy showed that SLN is a predominantly alpha-helical protein and that the secondary structure is highly resistant to SDS and thermal denaturation. In this respect SLN is remarkably similar to PLB-(24-52). However, SLN is monomeric in SDS gels, whereas PLB-(24-52) shows a monomer-pentamer equilibrium typical for native PLB. Analytical ultracentrifugation experiments revealed that SLN oligomerizes in the presence of the nonionic detergents octylpolyoxyethylene and octyl glucoside in a concentration-dependent manner. No plateau was observed, and a pentameric state was only reached at much higher protein concentrations compared with PLB-(24-52). Chemical cross-linking showed that also in liposomes SLN has the ability to self-associate to oligomers. PLB-(24-52) specifically oligomerized to pentamers in the presence of octylpolyoxyethylene as well as in liposomes at low protein concentrations. In the presence of octylpolyoxyethylene pentamers were the main oligomeric species, whereas in liposomes monomers and dimers were predominant. Increasing the protein concentration led to self-association of PLB-(24-52) pentamers in the presence of octylpolyoxyethylene. Functional reconstitution of Ca-ATPase with PLB-(24-52) and SLN in liposomes showed that both proteins regulate the Ca-ATPase in a similar manner.

Incorporation of beta-selenolo[3,2-b]pyrrolyl-alanine into proteins for phase determination in protein X-ray crystallography.

beta-Selenolo[3,2-b]pyrrolyl-L-alanine that mimics tryptophan with the benzene ring of the indole moiety replaced by selenophene, was incorporated into human annexin V and barstar. This was achieved by fermentation and expression in a Trp-auxotrophic Escherichia coli host strain using the selective pressure incorporation method. The seleno- proteins were obtained in yields comparable to those of the wild-type proteins and exhibit full crystallographic isomorphism to the parent proteins, but expectedly show altered absorbance profiles and quenched tryptophan fluorescence. Since the occurrence of tryptophan residues in proteins is rare, incorporation of the electron-rich selenium-containing tryptophan surrogate into proteins represents a useful supplementation and even a promising novel alternative to selenomethionine for solving the phase problem in protein X-ray crystallography.

Proteins with beta-(thienopyrrolyl)alanines as alternative chromophores and pharmaceutically active amino acids.

L-beta-(Thieno[3,2-b]pyrrolyl)alanine and L-beta-(thieno[2,3-b]pyrrolyl)alanine are mutually isosteric and pharmaceutically active amino acids that mimic tryptophan with the benzene ring in the indole moiety replaced by thiophene. Sulfur as a heteroatom causes physicochemical changes in these tryptophan surrogates that bring about completely new properties not found in the indole moiety. These synthetic amino acids were incorporated into recombinant proteins in response to the Trp UGG codons by fermentation in a Trp-auxotrophic Escherichia coli host strain using the selective pressure incorporation method. Related protein mutants expectedly retain the secondary structure of the native proteins but show significantly changed optical and thermodynamic properties. In this way, new spectral windows, fluorescence, polarity, thermodynamics, or pharmacological properties are inserted into proteins. Such an engineering approach by translational integration of synthetic amino acids with a priori defined properties, as shown in this study, proved to be a novel and useful tool for protein rational design.

Bivalent inhibition of human beta-tryptase.

BACKGROUND: Human beta-tryptase is a mast cell specific trypsin-like serine protease that is thought to play a key role in the pathogenesis of diverse allergic and inflammatory disorders like asthma and psoriasis. The recently resolved crystal structure revealed that the enzymatically active tetramer consists of four quasi-identical monomers. The spatial display of the four identical active sites represents an ideal basis for the rational design of bivalent inhibitors. RESULTS: Based on modeling experiments homobivalent inhibitors were constructed using (i) 6A,6D-dideoxy-6A,6D-diamino-beta-cyclodextrin as a rigid template to bridge the space between the two pairs of identical active sites and (ii) 3-(aminomethyl)benzene as a headgroup to occupy the arginine/lysine specific S1 subsites. A comparative analysis of the inhibitory potencies of synthetic constructs that differ in size and type of the spacer between headgroup and template revealed that the construct contained two 3-(aminomethyl)benzenesulfonyl-glycine groups linked to the 6A,6D-diamino groups of beta-cyclodextrin as an almost ideal bivalent inhibitor with a cooperativity factor of 1.9 vs. the ideal value of 2. The bivalent binding mode is supported by the inhibitor/tetramer ratio of 2:1 required for inactivation of tryptase and by X-ray analysis of the inhibitor/tryptase complex. CONCLUSION: The results obtained with the rigid cyclodextrin template underlined the importance of a minimal loss of conformational entropy in bivalent binding, but also showed the limitations imposed by such rigid core molecules in terms of optimal occupancy of binding sites and thus of enthalpic strains in bidentate binding modes. The main advantage of bivalent inhibitors is their high selectivity for the target enzyme that can be achieved utilizing the principle of multivalency.

The substrate translocation channel of the proteasome.

The core particle (CP) of the yeast proteasome is composed of four heptameric rings of subunits arranged in a hollow, barrel-like structure. We have found that the CP is autoinhibited by the N-terminal tails of the outer (alpha) ring subunits. Crystallographic analysis showed that deletion of the tail of the alpha3 subunit opens a channel into the proteolytically active interior chamber of the CP, thus derepressing peptide hydrolysis. In the latent state of the particle, the tails prevent substrate entry by imposing topological closure on the CP. Inhibition by the alpha subunit tails is relieved upon binding of the regulatory particle to the CP to form the proteasome holoenzyme. Opening of the CP channel by assembly of the holoenzyme is regulated by the ATPase domain of Rpt2, one of 17 subunits in the RP. Thus, open-channel mutations in CP subunits suppress the closed-channel phenotype of an rpt2 mutant. These results identify a specific mechanism for allosteric regulation of the CP by the RP.

Photoresponsive dendritic azobenzene peptides.

Two dendritic peptides containing a branched lysine core and up to eight azobenzene moieties in the periphery were synthesized on solid support employing the omega-amino acid 4-(aminomethyl)phenylazobenzoic acid. With an additional peptidic tail consisting of an oligolysine portion, water solubility was achieved for the dendrimers, which allowed for the characterization of the cis/trans photoisomerization of the dendritic azobenzene species in both organic and aqueous media. Despite the interactions between the chromophores, which occur particularly in aqueous media, at higher dilution the photoisomerization process was found to proceed to extents that should permit photomodulation of molecular recognition processes between ligands grafted to the photosensitive azobenzene units and receptor molecules.

The uPA/uPA receptor system as a target for tumor therapy.

Invasiveness of a variety of tumors depends on the regulated expression of proteolytic enzymes that degrade the surrounding extracellular matrix and dissociate cell-cell and/or cell-matrix attachments. The tumor cell surface-associated urokinase-type plasminogen activator (uPA) system plays an especially important role in tumor cell invasion and metastasis. It consists of the serine protease uPA, its membrane-bound receptor (uPAR, CD87) and one of the natural inhibitors PAI-1 or PAI-2. There are strong indications based on animal experiments that interference with this system by inhibiting the enzymatic activity of uPA and/or antagonizing its binding to the receptor is of therapeutic relevance. With the recent solution of various X-ray structures of uPA/inhibitor complexes, structural information is available for optimizing existing lead compounds in their affinity and selectivity for uPA. Furthermore, peptide compounds capable of mimicking the structural epitope of uPA responsible for binding to the receptor efficiently antagonize this recognition process. Thus, both approaches prove to be well suited for the design of highly promising drugs in human medicine.

Mutational studies on HslU and its docking mode with HslV.

HslVU is an ATP-dependent prokaryotic protease complex. Despite detailed crystal and molecular structure determinations of free HslV and HslU, the mechanism of ATP-dependent peptide and protein hydrolysis remained unclear, mainly because the productive complex of HslV and HslU could not be unambiguously identified from the crystal data. In the crystalline complex, the I domains of HslU interact with HslV. Observations based on electron microscopy data were interpreted in the light of the crystal structure to indicate an alternative mode of association with the intermediate domains away from HslV. By generation and analysis of two dozen HslU mutants, we find that the amidolytic and caseinolytic activities of HslVU are quite robust to mutations on both alternative docking surfaces on HslU. In contrast, HslVU activity against the maltose-binding protein-SulA fusion protein depends on the presence of the I domain and is also sensitive to mutations in the N-terminal and C-terminal domains of HslU. Mutational studies around the hexameric pore of HslU seem to show that it is involved in the recognition/translocation of maltose-binding protein-SulA but not of chromogenic small substrates and casein. ATP-binding site mutations, among other things, confirm the essential role of the "sensor arginine" (R393) and the "arginine finger" (R325) in the ATPase action of HslU and demonstrate an important role for E321. Additionally, we report a better refined structure of the HslVU complex crystallized along with resorufin-labeled casein.

A gated channel into the proteasome core particle.

The core particle (CP) of the yeast proteasome is composed of four heptameric rings of subunits arranged in a hollow, barrel-like structure. We report that the CP is autoinhibited by the N-terminal tails of the outer (alpha) ring subunits. Crystallographic analysis showed that deletion of the tail of the alpha 3-subunit opens a channel into the proteolytically active interior chamber of the CP, thus derepressing peptide hydrolysis. In the latent state of the particle, the tails prevent substrate entry by imposing topological closure on the CP. Inhibition by the alpha-subunit tails is relieved upon binding of the regulatory particle to the CP to form the proteasome holoenzyme.

Epoxysuccinyl peptide-derived affinity labels for cathepsin B.

Extracellular cysteine proteases, in particular cathepsin B, have been implicated in a variety of pathological processes. Selectively targeting labels of this enzyme are important tools to gain more detailed understanding of its specific roles. Starting from our recently developed irreversible epoxysuccinyl-based inhibitor (R-Gly-Gly-Leu-(2S,3S)-tEps-Leu-Pro-OH, R=OMe), we have synthesized two affinity labels, R=NH-(CH(2))(6)-NH-rhodamine B and R=NH-(CH(2))(6)-NH-biotin. Using MCF-7 cells, the labeled inhibitors were shown to be virtually non-cell-permeant. Moreover, affinity blot analysis with the biotinylated inhibitor allowed a highly sensitive and selective non-radioactive detection of active cathepsin B.