[Pediatric anesthesia for proton radiotherapy : medicine remote from the medical centre]. / Kinderanästhesie zur protonenbestrahlung : medizin fernab der klinik.

Anesthesia care for infants and young children for proton beam radiotherapy demands great technical and vocational skills from the anesthesia team and also a high degree of competence in soft skills. The anesthesia team should be experienced and regularly trained in pediatric anesthesia, especially as the children are often in a reduced general condition. The infrastructure should be established according to the current standards in anesthesiology. Monitoring of vital data, thorax excursions and inadvertent movements of the remotely positioned and sedated patient need to be under constant technical and optical surveillance. Propofol is an ideal hypnotic for the sedation of children under spontaneous breathing for proton beam radiation therapy. It is well tolerated even when given on a daily basis over several weeks. A close cooperation between the pediatric oncologist, radiation oncologist and anesthetist is important in order to manage additional medical problems in an optimal way. The special needs of oncology patients must be taken into consideration when planning anesthesia care.

Incidence and predictors of indwelling arterial catheter-related thrombosis in children.

BACKGROUND: Indwelling arterial catheters (IACs) are used for monitoring and blood sampling purposes in intensive care units. Very limited information is available on the incidence and risk factors of IAC-related thrombosis in children. OBJECTIVE: To investigate the incidence and predictors of IAC-related thrombosis in a tertiary care pediatric hospital. METHODS: For a period of 12 months, detailed information was prospectively recorded for all consecutive children requiring IACs. RESULTS: Six hundred and fifteen IACs were placed in a total of 473 children at a median age of 0.56 years for a total of 47440.84 catheter hours. Of the 615 IACs, 418 (68%) were placed in the radial artery, 137 (22%) in the femoral artery, 26 (4%) in the umbilical artery, 11 (2%) in the brachial artery, and 23 (3.7%) in another artery. Thrombosis occurred in 20 cases, reflecting an overall incidence of 3.25%. Eighteen of the 20 IAC-related thrombi were located in the femoral arteries, reflecting a relative incidence of 13% (18/137). Newborn age, lower body weight, low cardiac output and increased hematocrit were significantly related with an increased risk of femoral artery thrombosis. In logistic regression analysis, younger age (P<0.001, odds ratio 6.51) was independently associated with an increased thrombotic risk. CONCLUSIONS: This study demonstrates that arterial thrombosis occurs with an increased incidence in children requiring IACs in the femoral location. Younger age is independently associated with an increased risk of thrombosis. The radial location is safe, and should be preferred to the femoral location.

[Intraosseous infusion in the pediatric emergency medical service. Analysis of emergency medical missions 1990-2009]. / Einsatz der intraossären Infusion im pädiatrischen Notarztdienst. Analyse von Notarzteinsätzen 1990-2009.

BACKGROUND: Timely establishment of venous access in infants and toddlers during emergency medical care can be a particularly challenging task. Alternative routes for drug and fluid administration, such as endobronchial, intramuscular, central venous or venous cut-down do not offer reliable solutions. Intraosseous infusion (IOI) has become established as an effective alternative intravascular access for rapid and efficient drug delivery. IOI was introduced in our local emergency medical service (EMS) in 1993 and was assigned a high priority in international guidelines for pediatric emergency medical care in 2000 and 2005. The aim of this study was to review the impact of the introduction of IOI on drug administration routes during prehospital emergency treatment of critically ill or severely injured pediatric patients (NACA index V-VII) in our tertiary medical care centre over a period of 20 years. METHODS: Pediatric prehospital emergency medical protocols from 1990 to 2009 were analyzed with respect to the administration routes for fluids and medications in severely injured or critically ill children with NACA severity scores V-VII. The frequency and mode of vascular access during prehospital treatment including IOI, endobronchial administration, central venous catheterization (CVC) and intramuscular administration as well as prehospital treatment and transportation without vascular access were analyzed. Two groups were compared: the introduction phase of IOI between 1990 and 1999 and the phase of growing IOI routine after introducing guidelines and regular staff IOI technique training between 2000 and 2009. Demographic data and drug administration routes in the two different time periods were analyzed using the Mann-Whitney-u test and t-test or χ(2)-test, respectively. A p-value <0.05 was regarded as significant. RESULTS: A total of 5,279 pediatric prehospital emergency charts were analyzed and 401 patients (7.6%) were scored as NACA V-VII. At the emergency scene 299 patients (75%) received a peripheral intravenous access, 3 (0.7%) a central venous line access, 77 (19%) an intraosseous needle and in 22 (5.4%) no vascular or intraosseous access was used during the course of prehospital treatment (NACA VII - 13 patients, NACA VI - 2 patients, NACA V - 7 patients). Of the NACA VII patients 3 were transported under continuous cardiopulmonary resuscitation without vascular access. After 2002 all patients with NACA index VII were treated with vascular or intraosseous access. In 48 patients (12%) at least initial medication was given by the endobronchial or alternative route but within the last 3 years endobronchial drug administration was no longer reported. Thus, in 124 critically ill patients (31%) routine peripheral venous access could not be established initially or until the end of treatment (77 times IOI, 22 times no access over the course of treatment, 3 times CVC and 22 times initial endobronchial followed by peripheral venous access). Over the reviewed period the use of IOI increased significantly (p<0.001), while the incidence of lacking vascular access (p<0.05) and alternative drug administration routes (p<0.001) continuously decreased. CONCLUSION: The IOI technique has not only been assigned a high priority in the guidelines for pediatric emergency care of critically ill children with difficult or failed venous access but has also significantly influenced current prehospital care. The introduction of the IOI technique in our prehospital pediatric emergency system has markedly reduced the number of critically ill or severely injured pediatric patients without vascular access or with less reliable alternative administration routes in the last 20 years.

[Intraosseous infusion. An important technique also for paediatric anaesthesia]. / Intraossäre Infusion. Eine wichtige Technik auch für die Kinderanästhesie.

Timely establishment of venous access in infants and toddlers can prove a particularly challenging task. Since the 1940s the technique of intraosseous infusion has established itself as a valuable alternative means for rapid, efficient and safe delivery of drugs and fluids to critically ill children. Whereas international guidelines for paediatric emergency medical care have assigned intraosseous infusion a high priority, most anaesthetists utilize this well-proven technique with great reluctance. This article describes the technique of intraosseous infusion, introduces two different cannulation systems, and discusses its potential indications in paediatric anaesthesia, based on current emergency medical care guidelines as well as some of our own case studies. In particular, children with acutely life-threatening conditions, such as circulatory arrest, laryngospasm, acute airway haemorrhage, hypovolaemic shock or hypothermia secondary to extensive burns, should receive an intraosseous cannula if intravenous access cannot be rapidly established. Future discussion may reveal whether a transiently inserted intraosseous infusion would also be indicated if the child with difficult or impossible venous access presents without acute life-threatening conditions for anaesthesia. Successful application of the intraosseous infusion technique requires immediate access to the necessary equipment, intensive education, continuous training and clear guidelines for its application in an anaesthesia department.

HMG-D complexed to a bulge DNA: an NMR model.

An NMR model is presented for the structure of HMG-D, one of the Drosophila counterparts of mammalian HMG1/2 proteins, bound to a particular distorted DNA structure, a dA(2) DNA bulge. The complex is in fast to intermediate exchange on the NMR chemical shift time scale and suffers substantial linebroadening for the majority of interfacial resonances. This essentially precludes determination of a high-resolution structure for the interface based on NMR data alone. However, by introducing a small number of additional constraints based on chemical shift and linewidth footprinting combined with analogies to known structures, an ensemble of model structures was generated using a computational strategy equivalent to that for a conventional NMR structure determination. We find that the base pair adjacent to the dA(2) bulge is not formed and that the protein recognizes this feature in forming the complex; intermolecular NOE enhancements are observed from the sidechain of Thr 33 to all four nucleotides of the DNA sequence step adjacent to the bulge. Our results form the first experimental demonstration that when binding to deformed DNA, non-sequence-specific HMG proteins recognize the junction between duplex and nonduplex DNA. Similarities and differences of the present structural model relative to other HMG-DNA complex structures are discussed.

Solution structure of a C-terminal coiled-coil domain from bovine IF(1): the inhibitor protein of F(1) ATPase.

Bovine IF(1) is a basic, 84 amino acid residue protein that inhibits the hydrolytic action of the F(1)F(0) ATP synthase in mitochondria under anaerobic conditions. Its oligomerization state is dependent on pH. At a pH value below 6.5 it forms an active dimer. At higher pH values, two dimers associate to form an inactive tetramer. Here, we present the solution structure of a C-terminal fragment of IF(1) (44-84) containing all five of the histidine residues present in the sequence. Most unusually, the molecule forms an anti-parallel coiled-coil in which three of the five histidine residues occupy key positions at the dimer interface.

Solution studies of chymotrypsin inhibitor-2 glutamine insertion mutants show no interglutamine interactions.

Mutants of chymotrypsin inhibitor protein 2 have previously been studied in which 4 or 10 glutamine residues were inserted into the inhibitory loop of the protein between residues 59 and 60, as potential models for the behaviour of glutamine tracts in proteins associated with polyglutamine-expansion neurodegenarative diseases. These mutants form very stable monomers, dimers and trimers. Although the cause of oligomerisation was found to be domain-swapping, it was thought that the glutamine insertions might nevertheless show evidence of weak interglutamine interactions in solution that could mimic those occurring in disease-associated proteins. In the present NMR study, we used steady-state (15)N[(1)H] NOE measurements and chemical shift comparisons to characterise the motional properties of the inserted glutamines in these CI2 mutants. We found the glutamines to be highly mobile, with no evidence of interactions amongst them in either monomers or dimers.

The structural basis for the recognition of acetylated histone H4 by the bromodomain of histone acetyltransferase gcn5p.

The bromodomain is an approximately 110 amino acid module found in histone acetyltransferases and the ATPase component of certain nucleosome remodelling complexes. We report the crystal structure at 1.9 A resolution of the Saccharomyces cerevisiae Gcn5p bromodomain complexed with a peptide corresponding to residues 15-29 of histone H4 acetylated at the zeta-N of lysine 16. We show that this bromodomain preferentially binds to peptides containing an N:-acetyl lysine residue. Only residues 16-19 of the acetylated peptide interact with the bromodomain. The primary interaction is the N:-acetyl lysine binding in a cleft with the specificity provided by the interaction of the amide nitrogen of a conserved asparagine with the oxygen of the acetyl carbonyl group. A network of water-mediated H-bonds with protein main chain carbonyl groups at the base of the cleft contributes to the binding. Additional side chain binding occurs on a shallow depression that is hydrophobic at one end and can accommodate charge interactions at the other. These findings suggest that the Gcn5p bromodomain may discriminate between different acetylated lysine residues depending on the context in which they are displayed.

Growth and form of planetary seedlings: results from a microgravity aggregation experiment.

The outcome of the first stage of planetary formation, which is characterized by ballistic agglomeration of preplanetary dust grains due to Brownian motion in the free molecular flow regime of the solar nebula, is still somewhat speculative. We performed a microgravity experiment flown onboard the space shuttle in which we simulated, for the first time, the onset of free preplanetary dust accumulation and revealed the structures and growth rates of the first dust agglomerates in the young solar system. We find that a thermally aggregating swarm of dust particles evolves very rapidly and forms unexpected open-structured agglomerates.

The NMR structure of the 38 kDa U1A protein - PIE RNA complex reveals the basis of cooperativity in regulation of polyadenylation by human U1A protein.

The status of the poly(A) tail at the 3'-end of mRNAs controls the expression of numerous genes in response to developmental and extracellular signals. Poly(A) tail regulation requires cooperative binding of two human U1A proteins to an RNA regulatory region called the polyadenylation inhibition element (PIE). When bound to PIE RNA, U1A proteins also bind to the enzyme responsible for formation of the mature 3'-end of most eukaryotic mRNAs, poly(A) polymerase (PAP). The NMR structure of the 38 kDa complex formed between two U1A molecules and PIE RNA shows that binding cooperativity depends on helix C located at the end of the RNA-binding domain and just adjacent to the PAP-interacting domain of U1A. Since helix C undergoes a conformational change upon RNA binding, the structure shows that binding cooperativity and interactions with PAP occur only when U1A is bound to its cognate RNA. This mechanism ensures that the activity of PAP enzyme, which is essential to the cell, is only down regulated when U1A is bound to the U1A mRNA.

Determination of the NMR structure of the complex between U1A protein and its RNA polyadenylation inhibition element.

RNA-protein recognition is critical to post-transcriptional regulation of gene expression, yet poorly understood at the molecular level. The relatively slow progress in understanding this important area of molecular biology is due to difficulties in obtaining good-quality crystals and derivatives, and in preparing samples suitable for NMR investigation. The determination of the structure of the complex between the human U1A protein and its polyadenylation inhibition element is described here. In this paper, we describe the sample preparation, spectral assignments, construction of the NOE-based distance constraints and methodology for calculating the structure of the complex. The structure was determined to an overall precision of 2.03 A (for all ordered regions), and 1.08 A for the protein-RNA interface. The patterns of hydrogen bonding and hydrophobic interactions at the interface were analysed statistically using the final ensemble of 31 structures.

Solution structure of the motile major sperm protein (MSP) of Ascaris suum - evidence for two manganese binding sites and the possible role of divalent cations in filament formation.

The major sperm protein (MSP) of Ascaris suum mediates amoeboid motility by forming an extensive intermeshed system of cytoskeletal filaments analogous to that formed by actin in many other amoeboid cells. MSP is a dimeric molecule that polymerizes to form non-polar filaments constructed from two helical subfilaments that wind round one another. Moreover, MSP filaments can interact with one another to form higher-order assemblies without requiring the range of accessory proteins usually employed in actin-based systems. A knowledge of how MSP polymerizes and forms the hierarchical series of helical MSP macromolecular assemblies is fundamental to understanding locomotion in these cells. Here we describe the solution structure of MSP dimers determined by NMR spectroscopy under conditions where MSP does not polymerize to form filaments. The solution structure is indistinguishable from that observed in putative MSP subfilament helices by X-ray crystallography, indicating that MSP polymerization is not accompanied by a major conformational change. We also show that the rate of MSP polymerization associated with movement of vesicles in an in vitro motility assay is enhanced by the presence of magnesium and manganese ions and use NMR to show that the primary residues that bind these ions are 24-25 and 83-86. These residues are distant from the interface formed between MSP dimers in subfilament helices, and so are probably not involved in this level of polymerization. Instead the manganese and magnesium ion binding appears to be associated with the assembly of subfilaments into filaments and their subsequent aggregation into bundles.

Structural basis of the RNA-binding specificity of human U1A protein.

The RNP domain is a very common eukaryotic protein domain involved in recognition of a wide range of RNA structures and sequences. Two structures of human U1A in complex with distinct RNA substrates have revealed important aspects of RNP-RNA recognition, but have also raised intriguing questions concerning the origin of binding specificity. The beta-sheet of the domain provides an extensive RNA-binding platform for packing aromatic RNA bases and hydrophobic protein side chains. However, many interactions between functional groups on the single-stranded nucleotides and residues on the beta-sheet surface are potentially common to RNP proteins with diverse specificity and therefore make only limited contribution to molecular discrimination. The refined structure of the U1A complex with the RNA polyadenylation inhibition element reported here clarifies the role of the RNP domain principal specificity determinants (the variable loops) in molecular recognition. The most variable region of RNP proteins, loop 3, plays a crucial role in defining the global geometry of the intermolecular interface. Electrostatic interactions with the RNA phosphodiester backbone involve protein side chains that are unique to U1A and are likely to be important for discrimination. This analysis provides a novel picture of RNA-protein recognition, much closer to our current understanding of protein-protein recognition than that of DNA-protein recognition.

Paradoxical sphincter contraction is rarely indicative of anismus.

BACKGROUND: Anismus is thought to be a cause of chronic constipation by producing outlet obstruction. The underlying mechanism is paradoxical contraction of the anal sphincter or puborectalis muscle. However, paradoxical sphincter contraction (PSC) also occurs in healthy controls, so anismus may be diagnosed too often because it may be based on a non-specific finding related to untoward conditions during the anorectal examination. AIMS: To investigate the pathophysiological importance of PSC found at anorectal manometry in constipated patients and in patients with stool incontinence. METHODS: Digital rectal examination and anorectal manometry were performed in 102 chronically constipated patients, 102 patients with stool incontinence, and in 18 controls without anorectal disease. In 120 of the 222 subjects defaecography was also performed. Paradoxical sphincter contraction was defined as a sustained increase in sphincter pressure during straining. Anismus was assumed when PSC was present on anorectal manometry and digital rectal examination and the anorectal angle did not widen on defaecography. RESULTS: Manometric PSC occurred about twice as often in constipated patients as in incontinent patients (41.2% versus 25.5%, p < 0.017) and its prevalence was similar in incontinent patients and controls (25.5% versus 22.2%). Oroanal or rectosigmoid transit times in constipated patients with and without PSC did not differ significantly (total 64.6 (8.9) hours versus 54.2 (8.1) hours; rectosigmoid 14.9 (2.4) hours versus 13.8 (2.5) hours). CONCLUSIONS: Paradoxical sphincter contraction is a common finding in healthy controls as well as in patients with chronic constipation and stool incontinence. Hence, PSC is primarily a laboratory artefact and true anismus is rare.

The motile major sperm protein (MSP) from Ascaris suum is a symmetric dimer in solution.

The major sperm protein (MSP) of Ascaris suum mediates amoeboid motility by forming an extensive intermeshed system of cytoskeletal filaments analogous to that formed by actin in many amoeboid cells. We have used a combination of biochemical and NMR methods to show that, in contrast to actin, MSP exist in solution as a symmetrical dimer. This result has important implications for the mechanism of both MSP filament assembly and the recognition of different MSP isoforms in vivo.

The solution structure of the first zinc finger domain of SWI5: a novel structural extension to a common fold.

BACKGROUND: The 2Cys-2His (C2-H2) zinc finger is a protein domain commonly used for sequence-specific DNA recognition. The zinc fingers of the yeast transcription factors SWI5 and ACE2 share strong sequence homology, which extends into a region N-terminal to the first finger, suggesting that the DNA-binding domains of these two proteins include additional structural elements. RESULTS: Structural analysis of the zinc fingers of SWI5 reveals that a 15 residue region N-terminal to the finger motifs forms part of the structure of the first finger domain, adding a beta strand and a helix not previously observed in other zinc finger structures. Sequence analysis suggests that other zinc finger proteins may also have this structure. Biochemical studies show that this additional structure increases DNA-binding affinity. CONCLUSIONS: The structural analysis presented reveals a novel zinc finger structure in which additional structural elements have been added to the C2-H2 zinc finger fold. This additional structure may enhance stability and has implications for DNA recognition by extending the potential DNA-binding surface of a single zinc finger domain.

Specificity of ribonucleoprotein interaction determined by RNA folding during complex formulation.

Many proteins involved in pre-mRNA processing contain one or more copies of a 70-90-amino-acid alphabeta module called the ribonucleoprotein domain. RNA maturation depends on the specific recognition by ribonucleoproteins of RNA elements within pre-mRNAs and small nuclear RNAs. The human U1A protein binds an RNA hairpin during splicing, and regulates its own expression by binding an internal loop in the 3'-untranslated region of its pre-mRNA, preventing polyadenylation. Here we report the nuclear magnetic resonance structure of the complex between the regulatory element of the U1A 3'-untranslated region (UTR) and the U1A protein RNA-binding domain. Specific intermolecular recognition requires the interaction of the variable loops of the ribonucleoprotein domain with the well-structured helical regions of the RNA. Formation of the complex then orders the flexible RNA single-stranded loop against the protein beta-sheet surface, and reorganizes the carboxy-terminal region of the protein to maximize surface complementarity and functional group recognition.

Solution structure of the N-terminal RNP domain of U1A protein: the role of C-terminal residues in structure stability and RNA binding.

The solution structure of a fragment of the human U1A spliceosomal protein containing residues 2 to 117 (U1A117) determined using multi-dimensional heteronuclear NMR is presented. The C-terminal region of the molecule is considerably more ordered in the free protein than thought previously and its conformation is different from that seen in the crystal structure of the complex with U1 RNA hairpin II. The residues between Asp90 and Lys98 form an alpha-helix that lies across the beta-sheet, with residues IIe93, IIe94 and Met97 making contacts with Leu44, Phe56 and IIe58. This interaction prevents solvent exposure of hydrophobic residues on the surface of the beta-sheet, thereby stabilising the protein. Upon RNA binding, helix C moves away from this position, changing its orientation by 135 degrees to allow Tyr13, Phe56 and Gln54 to stack with bases of the RNA, and also allowing Leu44 to contact the RNA. The new position of helix C in the complex with RNA is stabilised by hydrophobic interactions from IIe93 and IIe94 to IIe58, Leu 41, Val62 and His 10, as well as a hydrogen bond between Ser91 and Thr11. The movement of helix C mainly involves changes in the main-chain torsion angles of Thr89, Asp90 and Ser91, the helix thereby acting as a "lid" over the RNA binding surface.

Treatment of NOE constraints involving equivalent or nonstereoassigned protons in calculations of biomacromolecular structures.

Two modifications to the commonly used protocols for calculating NMR structures are developed, relating to the treatment of NOE constraints involving groups of equivalent protons or nonstereoassigned diastereotopic protons. Firstly, a modified method is investigated for correcting for multiplicity, which is applicable whenever all NOE intensities are calibrated as a single set and categorised in broad intensity ranges. Secondly, a new set of values for 'pseudoatom corrections' is proposed for use with calculations employing 'centre-averaging'. The effect of these protocols on structure calculations is demonstrated using two proteins, one of which is well defined by the NOE data, the other less so. It is shown that failure to correct for multiplicity when using 'r(-6) averaging' results in overly precise structures, higher NOE energies and deviations from geometric ideality, while failure to correct for multiplicity when using 'r(-6) summation' can cause an avoidable degradation of precision if the NOE data are sparse. Conversely, when multiplicities are treated correctly, r(-6) averaging, r(-6) summation and centre averaging all give closely comparable results when the structure is well defined by the data. When the NOE data contain less information, r(-6) averaging or r(-6) summation offer a significant advantage over centre averaging, both in terms of precision and in terms of the proportion of calculations that converge on a consisten result.