Perceptual type error in everyday practice.

Discrepancy or quality improvement meetings are good practice and are now commonplace in most Radiology Departments, with the aim of improving diagnostic accuracy, preventing recurrent and common mistakes, improving the radiological report and thereby improving patient care. A total of 122 cases were assessed from a two-year period. This review highlights some of the more common, recurrent and important issues encountered within a general hospital with an emphasis on learning points and review areas.

Imaging submandibular pathology in the paediatric patient.

A wide range of pathologies may arise from the submandibular space (SMS) or submandibular gland (SMG) in children. We review herein the normal anatomy of the SMS and describe the role of imaging in the evaluation of SMS lesions. A schematic approach for the categorisation of SMS pathology based on imaging characteristics is provided.

Differential laser absorption spectroscopy of uranium in an atmospheric pressure laser-induced plasma.

A two-beam differential laser absorption technique is used to measure 238U absorption spectra with high signal-to-noise ratios in an atmospheric pressure laser-induced plasma. High-resolution absorption spectra are presented for the 238U 861 nm transition in the presence of dry air at pressures up to 760 Torr. A spectral linewidth (FWHM) of 2.23±0.13 GHz was found for the 238U line in dry air at 760 Torr. Absorption spectrum measurements using a low 238U concentration NIST glass standard were used to demonstrate sensitivity of the approach.

Hard-wired models of working memory and temporal sequence storage and generation.

We develop hard-wired simulations of temporal sequence storage and generation by multi-modular networks based on the frontal lobe system (cortex, basal ganglia and thalamus). Single cell activity is shown to have been constructed so as to mimic results measured in monkeys on a similar task, indicating that a suitable form of chunking had been achieved in the models. The mathematical nature of these processes is discussed, from the viewpoint of bifurcation theory.

Analysis of recurrent cortico-basal ganglia-thalamic loops for working memory.

We analyse a simplified form of the frontal lobe architecture of cortico-basal ganglia-thalamo-cortical loops to determine the manner in which they can learn temporal sequences as part of working memory activity. In particular, we consider how the temporal duration of activity can arise in this setting. We start from a hard-wired version in which temporally extended activity is created by the 'long' loop of cortex --> basal ganglia --> thalamus --> cortex, and show it arises from a near saddle-node bifurcation. The manner in which the transition between patterns occurs is also considered. This is then extended to analyse the temporal sequence storage and regeneration abilities of trained networks with a similar architecture. The temporal dynamics of this activity is also analysed. Implications of this for other working memory activities and for understanding the architecture of the frontal lobes are discussed in conclusion.

Isolated cleft palate in mice with a targeted mutation of the LIM homeobox gene lhx8.

Formation of the mammalian secondary palate is a highly regulated and complex process whose impairment often results in cleft palate, a common birth defect in both humans and animals. Loss-of-function analysis has linked a growing number of genes to this process. Here we report that Lhx8, a recently identified LIM homeobox gene, is expressed in the mesenchyme of the mouse palatal structures throughout their development. To test the function of Lhx8 in vivo, we generated a mutant mouse with a targeted deletion of the Lhx8 gene. Our analysis of the mutant animals revealed a crucial role for Lhx8 in palatogenesis. In Lhx8 homozygous mutant embryos, the bilateral primordial palatal shelves formed and elevated normally, but they often failed to make contact and to fuse properly, resulting in a cleft secondary palate. Because development of other craniofacial structures appeared normal, the impaired palatal formation in Lhx8-mutant mice was most likely caused by an intrinsic primary defect in the mesenchyme of the palatal shelves. The cleft palate phenotype observed in Lhx8-mutant mice suggests that Lhx8 is a candidate gene for the isolated nonsyndromic form of cleft palate in humans.

Dihydropyrancarboxamides related to zanamivir: a new series of inhibitors of influenza virus sialidases. 2. Crystallographic and molecular modeling study of complexes of 4-amino-4H-pyran-6-carboxamides and sialidase from influenza virus types A and B.

The first paper in this series (see previous article) described structure-activity studies of carboxamide analogues of zanamivir binding to influenza virus sialidase types A and B and showed that inhibitory activity of these compounds was much greater against influenza A enzyme. To understand the large differences in affinities, a number of protein-ligand complexes have been investigated using crystallography and molecular dynamics. The crystallographic studies show that the binding of ligands containing tertiary amide groups is accompanied by the formation of an intramolecular planar salt bridge between two amino acid residues in the active site of the enzyme. It is proposed that the unexpected strong binding of these inhibitors is a result of the burial of hydrophobic surface area and salt-bridge formation in an environment of low dielectric. In sialidase from type A virus, binding of the carboxamide moeity and salt-bridge formation have only a minor effect on the positions of the surrounding residues, whereas in type B enzyme, significant distortion of the protein is observed. The results suggest that the decreased affinity in enzyme from influenza B is directly correlated with the small changes that occur in the amino acid residue interactions accompanying ligand binding. Molecular dynamics calculations have shown that the tendency for salt-bridge formation is greater in influenza A sialidase than influenza B sialidase and that this tendency is a useful descriptor for the prediction of inhibitor potency.

The World Wide Web as a graphical user interface to program macros for molecular graphics, molecular modeling, and structure-based drug design.

In this article we describe how the World Wide Web (WWW or Web) has been employed to provide access to computational chemistry software and protein structure data via program macros. We show how the combination of Web technology and macros can automate both the running of chemistry software and the execution of complex operations on protein structures. The current version of the system supports the molecular visualization packages GRASP, RASMOL, MOLVIEWER-OGL and INSIGHT95, and the ligand design tool GRID and includes more than 175 in-house protein-ligand complexes. The approach enables inexperienced users to confidently make full use of sophisticated modeling techniques by offering only sensible options, hiding parameter settings, and controlling program invocation and macro execution. Our interface provides both the expert and non-expert alike with powerful tools for protein structure visualization, molecular modeling, and rational drug design.

A structural and energetics analysis of the binding of a series of N-acetylneuraminic-acid-based inhibitors to influenza virus sialidase.

A molecular dynamics/energy-minimisation protocol has been used to analyse the structural and energetic effects of functional group substitution on the binding of a series of C4-modified 2-deoxy-2,3-didehydro-N-acetylneuraminic acid inhibitors to influenza virus sialidase. Based on the crystal structure of sialidase, a conformational searching protocol, incorporating multiple randomisation steps in a molecular dynamics simulation was used to generate a range of minimum-energy structures. The calculations were useful for predicting the number, location, and orientation of structural water molecules within protein-ligand complexes. Relative binding energies were calculated for the series of complexes using several empirical molecular modelling approaches. Energies were computed using molecular-mechanics-derived interactions as the sum of pairwise atomic nonbonded energies, and in a more rigorous manner including solvation effects as the change in total electrostatic energy of complexation, using a continuum-electrostatics (CE) approach. The CE approach exhibited the superior correlation with observed affinities. Both methods showed definite trends in observed and calculated binding affinities; in both cases inhibitors with a positively charged C4 substituent formed the tightest binding to the enzyme, as observed experimentally.

Anionic phospholipids bind to L-selectin (but not E-selectin) at a site distinct from the carbohydrate-binding site.

It is known that L-selectin binds to glycoconjugates containing the tetrasaccharide sialyl Lewis X in a Ca2+-dependent manner. In addition, a number of other acidic oligosaccharides (for example heparin or chondroitin sulphate) or glycolipids (for example sulphatides) bind to L-selectin independent of cations. In this paper we have established that L-selectin binds to charged phospholipids, such as cardiolipin and phosphatidylserine, but not to neutral phospholipids such as phosphatidylcholine. No interaction between E-selectin and any phospholipid was observed. The interaction between L-selectin cardiolipin was inhibited by dextran sulphate, fucoidan, mannose 6-phosphate and monoclonal antibodies previously reported to block the interaction between L-selectin and its natural ligands. Analysis of the amino acid sequence of the selectins indicated that L-selectin, but no E-selectin, contains a sequence homologous to the putative cardiolipin-binding epitope found in plasma glycoprotein beta2I. Glycoprotein beta2I and a peptide corresponding to the putative cardiolipin-binding epitope in beta2I inhibited the binding of L-selectin to cardiolipin or fucoidin. Based on the binding characteristics, sequence analysis and structural modelling of L-selectin, we suggest that the amino acid sequence KKNKED (residues 84-89) is a novel site for the binding of acidic species to L-selectin. This motif is localized close to the putative carbohydrate-binding domain of L-selectin and may be a second site within the lectin domain for the interaction of leucocyte L-selectin with its natural endothelial ligands.

New spiropiperidines as potent and selective non-peptide tachykinin NK2 receptor antagonists.

The synthesis of a series of 2-(5-fluoro-1H-indol-3-yl)ethyl spiropiperidines is described together with their tachykinin NK2 receptor affinities measured in a rat colon binding assay. Equivalent NK2 receptor binding affinity was observed for the spirooxazolidinone 3-benzyl-8-[2-(5-fluoro-1H-indol-3-yl)ethyl]-1-oxa-3,8-diazaspiro[4.5] decan-2-one (3a), the imidazolidinone 3-benzyl-8-[2-(5-fluoro-1H-indol-3-yl)ethyl]-1,3,8-triazaspiro[4.5 ] decan-2-one (3s), and the pyrrolidinone 2-benzyl-8-[2-(5-fluoro-1H-indol-3-yl)ethyl]-2,8-diazaspiro[4.5]decan -3 - one (3t). Substitution in the phenyl ring of compound 3a produced no significant enhancement in NK2 binding affinity. Replacement of the phenyl ring in 3a with other aromatic rings resulted in a significant loss in binding affinity. Compound 3a was shown to be a potent NK2 receptor antagonist in guinea pig trachea where it also demonstrated 1000-fold selectivity for NK2 receptors over NK1. In the anesthetized guinea pig, compound 3a administered by the intravenous or oral route displayed potent and long-lasting antagonist activity against NK2 receptor agonist induced bronchoconstriction.

Molecular modeling studies on ligand binding to sialidase from influenza virus and the mechanism of catalysis.

A molecular modeling study has been used to investigate the structural and energetic aspects of substrate and inhibitor binding and the mechanism of catalysis of influenza virus sialidase. A detailed analysis of the interactions of both N-acetylneuraminic acid (Neu5Ac,1) and a number of transition-state analogues with the active site of influenza A sialidase at an atomic level is reported. In each case the calculated structures favorably agreed with the results from X-ray studies. A qualitative agreement between the calculated binding energies for inhibitors with positive substituents at the C4 position on the sugar ring and experimental Ki values was observed. We propose that the hydrolysis of sialosides occurs via an SN1 type mechanism that is facilitated through an activated solvent water molecule which can be expelled upon inhibitor binding. A reaction scheme is presented that is consistent with previously observed crystallographic structures, anomeric products, and isotope effects.

Chimeric DNA-RNA hammerhead ribozymes have enhanced in vitro catalytic efficiency and increased stability in vivo.

Subsequent to the discovery that RNA can have site specific cleavage activity, there has been a great deal of interest in the design and testing of trans-acting catalytic RNAs as both surrogate genetic tools and as therapeutic agents. We have been developing catalytic RNAs or ribozymes with target specificity for HIV-1 RNA and have been exploring chemical synthesis as one method for their production. To this end, we have chemically synthesized and experimentally analyzed chimeric catalysts consisting of DNA in the non-enzymatic portions, and RNA in the enzymatic core of hammerhead type ribozymes. Substitutions of DNA for RNA in the various stems of a hammerhead ribozyme have been analyzed in vitro for kinetic efficiency. One of the chimeric ribozymes used in this study, which harbors 24 bases of DNA capable of base-pairing interactions with an HIV-1 gag target, but maintains RNA in the catalytic center and in stem-loop II, has a sixfold greater kcat value than the all RNA counterpart. This increased activity appears to be the direct result of enhanced product dissociation. Interestingly, a chimeric ribozyme in which stem-loop II (which divides the catalytic core) is comprised of DNA, exhibited a marked reduction in cleavage activity, suggesting that DNA in this region of the ribozyme can impart a negative effect on the catalytic function of the ribozyme. DNA-RNA chimeric ribozymes transfected by cationic liposomes into human T-lymphocytes are more stable than their all-RNA counterparts. Enhanced catalytic turnover and stability in the absence of a significant effect on Km make chimeric ribozymes favorable candidates for therapeutic agents.

Evidence for a sialosyl cation transition-state complex in the reaction of sialidase from influenza virus.

The enzyme mechanism of sialidase from influenza virus has been investigated by kinetic isotope methods, NMR, and a molecular dynamics simulation of the enzyme-substrate complex. Comparison of the reaction rates obtained with the synthetic substrate 4-methylumbelliferyl-N-acetyl-alpha-D-neuraminic acid and the [3,3-2H]-substituted substrate revealed beta-deuterium isotope effects for V/Km ranging over 1.09-1.15 in the pH range 6.0-9.5, whereas the effects observed for V in this pH range increased from 0.979 to 1.07. In D2O, beta DV/Km was slightly increased by 2% and 5% at pD 6.0 and 9.5 respectively, while beta DV was unchanged. Solvent isotope effects of 1.74 were obtained for both beta DV/Km and beta DV at pD 9.5, with beta DV/Km decreasing and beta DV remaining constant at acidic pD. 1H-NMR experiments confirmed that the initial product of the reaction is the alpha-anomer of N-acetyl-D-neuraminic acid. Molecular dynamics studies identified a water molecule in the crystal structure of the sialidase-N-acetyl-D-neuraminic acid complex which is hydrogen-bonded to Asp151 and is available to act as a proton donor source in the enzyme reaction. The results of this study lead us to propose a mechanism for the solvent-mediated hydrolysis of substrate by sialidase that requires the formation of an endocyclic sialosyl cation transition-state intermediate.

Ribozyme-mediated cleavage of an HIV-1 gag RNA: the effects of nontargeted sequences and secondary structure on ribozyme cleavage activity.

Catalytic antisense RNAs, or ribozymes, have great potential as inhibitors of gene expression and as antiviral therapeutic agents. The major advantage of ribozymes versus standard antisense RNAs is their catalytic capability, enabling these RNAs to cleave multiple substrates. We have been investigating the antiviral activity of ribozymes targeted to the HIV-1 genome. The successful use of these antisense agents in an intracellular milieu requires stabilization of the ribozymes by flanking, non-base-pairing sequences, or some modification of the sugar-phosphate backbone. We describe a systematic investigation of the effects of flanking, non-base-pairing sequences on the catalytic activity of an anti-HIV-1 gag ribozyme embedded in radically different transcripts. Amazingly, these complex ribozyme-containing transcripts maintain substantial catalytic activity. Finally, we describe a bacterial gene fusion system that has potential for the large scale production of catalytically active ribozymes.

Pseudotumor resulting from atypical mycobacterial infection: a "histoid" variety of Mycobacterium avium-intracellulare complex infection.

A 54-year-old immunosuppressed cardiac transplant recipient with a six-month history of progressive swelling of the hand, with nodules and linear lymph node chain enlargement, diagnosed as a sporotrichoid Mycobacterium avium-intracellulare pseudotumor is described. The microscopic features closely resembled the previously described histoid variety of lepromatous leprosy. Routine hematoxylin and eosin staining suggested a spindle cell neoplasm rather than an infectious or inflammatory process. An infectious etiology was pursued on the basis of the clinical setting.