Antimicrobial breakpoints-definitions and conflicting requirements.
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Animals↗
Biomedical subjects
R Bywater
Publications and source records attributed to R Bywater.
Surveillance of antimicrobial resistance--what, how and whither?
OBJECTIVE: To express the views of a working party held to consider antibiotic resistance surveillance systems, their strengths and weaknesses, and their current and future applications. METHODS: The participants, all of whom were experienced in this field, discussed the development of surveillance systems in relation to the increasing prevalence of resistance to antibacterial agents and the current interest in surveillance systems shown by many official bodies, in both the human and veterinary fields. The problems inherent in surveillance systems were considered together with the applications of different systems. RESULTS: The properties of good antibiotic resistance surveillance systems were defined. Surveillance systems vary widely from those with a narrow base, focusing on few organisms in one disease area, to those covering many diseases, many organisms (including normal flora) and many compounds. Whatever their design, they should be able to detect significant differences and shifts in susceptibility to various antibacterial agents, and the information derived from them should reach as many interested parties as possible in a timely manner. In using this information to decide strategies, criteria for action need to be determined by pragmatic consensus. Funding remains a major problem, with few large studies being supported by official bodies in spite of their professed enthusiasm for surveillance. In consequence, many current systems are funded by the pharmaceutical industry and are of necessity restricted in their focus. CONCLUSIONS: Antibiotic resistance surveillance studies should and can be well planned and well executed. Many current systems suffer from well-recognized but uncorrected biases. Consortium funding will be necessary for large schemes to be successful. There is no "ideal" surveillance system.
Animals↗
The interaction of class B G protein-coupled receptors with their hormones.
In common with many G protein-coupled receptors, dysfunction in members of the Class B or glucagon-like receptors can elicit a wide spectrum of disease related activities. Consequently, they are potential targets in many different areas of pharmacological research. Unlike the class A or rhodopsin-like receptors, for which at least some structural similarity to bacteriorhodopsin has been detected, absolutely no structural information is available for the Class B G protein-coupled receptors. We present a computational study that exploits the experimental work performed by evolution to indicate residues that are potentially involved in ligand binding in the Class B G protein-coupled receptors. We perform an analysis of mutations that occurred in a correlated fashion between the receptors and their peptidic ligands. The inference that the residues detected in this manner are involved in a direct interaction between the receptor and the ligand is in good agreement with the mutation studies that have already been published.
Amino Acid Sequence↗
Essential dynamics of lipase binding sites: the effect of inhibitors of different chain length.
The biochemical activity of enzymes, such as lipases, is often associated with structural changes in the enzyme resulting in selective and stereospecific reactions with the substrate. To investigate the effect of a substrate and its chain length on the dynamics of the enzyme, we have performed molecular dynamics simulations of the native Rhizomucor miehei lipase (Rml) and lipase-dialkylphosophate complexes, where the length of the alkyl chain ranges from two to 10 carbon atoms. Simulations were performed in water and trajectories of 400 ps were used to analyse the essential motions in these systems. Our results indicate that the internal motions of the Rml and Rml complexes occur in a subspace of only a few degrees of freedom. A high flexibility is observed in solvent-exposed segments, which connect beta-sheets and helices. In particular, loop regions Gly35-Lys50 and Thr57-Asn63 fluctuate extensively in the native enzyme. Upon activation and binding of the inhibitor, involving the displacement of the active site loop, these motions are considerably suppressed. With increasing chain length of the inhibitor, the fluctuations in the essential subspace increase, levelling off at a chain length of 10, which corresponds to the size of the active-site groove.
Amino Acid Sequence↗
PRODRG, a program for generating molecular topologies and unique molecular descriptors from coordinates of small molecules.
A software package is described that operates on small molecules observed in the PDB collection of protein structures. Molecular topology files for many molecular modeling programs can be generated automatically. The three-dimensional coordinates of small molecules can be converted to molecular descriptor strings that encode them uniquely in order to enable small-molecule recognition, despite high variability in atom and molecule nomenclature. From this descriptor a plausible 3D structure can be regenerated using energy minimisation. Alternatively, an ensemble of structures can be generated using a distance-geometry-based algorithm.
Ligands↗
Dynamics of proteins in different solvent systems: analysis of essential motion in lipases.
We have investigated the effect of different solvents on the dynamics of Rhizomucor miehei lipase. Molecular dynamics simulations were performed in water, methyl hexanoate, and cyclohexane. Analysis of the 400-ps trajectories showed that the solvent has a pronounced effect on the geometrical properties of the protein. The radius of gyration and total accessibility surface decrease in organic solvents, whereas the number of hydrogen bonds increases. The essential motions of the protein in different solvents can be described in a low-dimensional "essential subspace," and the dynamic behavior in this subspace correlates with the polarity of the solvent. Methyl hexanoate, which is a substrate for R. miehei lipase, significantly increases the fluctuations in the active-site loop. During the simulation, a methyl hexanoate entered the active-site groove. This observation provides insight into the possible docking mechanism of the substrate.
Fungal Proteins↗
A comparison of structural and dynamic properties of different simulation methods applied to SH3.
The dynamic and static properties of molecular dynamics simulations using various methods for treating solvent were compared. The SH3 protein domain was chosen as a test case because of its small size and high surface-to-volume ratio. The simulations were analyzed in structural terms by examining crystal packing, distribution of polar residues, and conservation of secondary structure. In addition, the "essential dynamics" method was applied to compare each of the molecular dynamics trajectories with a full solvent simulation. This method proved to be a powerful tool for the comparison of large concerted atomic motions in SH3. It identified methods of simulation that yielded significantly different dynamic properties compared to the full solvent simulation. Simulating SH3 using the stochastic dynamics algorithm with a vacuum (reduced charge) force field produced properties close to those of the full solvent simulation. The application of a recently described solvation term did not improve the dynamic properties. The large concerted atomic motions in the full solvent simulation as revealed by the essential dynamics method were analyzed for possible biological implications. Two loops, which have been shown to be involved in ligand binding, were seen to move in concert to open and close the ligand-binding site.
Algorithms↗
Desorption of immunoglobulins from Protein A-Sepharose CL-4B under mild conditions.
Conditions which permit the dissociation of IgG-staphylococcal protein A interactions without resort to low pH buffers or the use of chaotropic ions are discussed in relation to: (a) the mechanism of the binding reaction; (b) the use of immobilised protein A for purification of cells or removal of immune complexes from serum. Glycyl-tyrosine, glycyl-histidine, glycyl-phenylalanine and tryptophan, representing the class of competing ligand desorbents, and ethylene glycol, an agent which disrupts hydrophobic interactions, were found to be the best desorbents.
Animals↗
A novel chromatographic procedure for purification of bacterial plasmids.
A new, rapid procedure for purifying bacterial plasmids with high recovery is described. The sequence of operations consists essentially of treatment with alkali, ribonuclease, and proteinase K, followed by chisam extraction and gel filtration on Sephacryl S-1000, and finally a precipitation step using isopropanol at room temperature. The method gives rather good yields of plasmid DNA of high purity, and lends itself to scaling up.
Bacteria↗
The synthesis and characterisation of a cyclic AMP-Sepharose 4B matrix with affinity for cyclic AMP-dependent proteins.
A novel synthesis route for the preparation of a cyclic AMP-containing affinity adsorbent is described. This involves the use of a simple single-vessel, two-step, solid-phase reaction in which 5'-AMP, originally coupled to the matrix in the form of 5'-AMP-Sepharose 4B, is cyclised by a sequence involving activation of the phosphate moiety, followed by intramolecular condensation to give a 3',5'-phosphodiester. Physicochemical evidence, including 31P NMR studies, shows that this cyclisation takes place leaving no trace of original phosphomonoesters. The gel retains its excellent chromatographic properties, but now its specificity is directed towards cAMP-dependent proteins. Protein kinase (EC 2.7.1.37) binds to the gel, the catalytic subunit only being eluted when a simple buffer 0.2 M phosphate/1 mM theophylline is applied to column, whilst the regulatory unit is eluted using 20% ethylene glycol of 2% Pharmalyte, pH 5-8. This gel should find a use in the purification of protein kinase subunits and, by inference, of other cAMP-dependent proteins.
Adrenal Cortex↗
Improving the odds in discriminating "drug-like" from "non drug-like" compounds.
We have used a feed-forward neural network technique to classify chemical compounds into potentially "drug-like" and "non drug-like" candidates. The neural network was trained to distinguish between a set of "drug-like" and "non drug-like" chemical compounds taken from the MACCS-II Drug Data Report (MDDR) and the Available Chemicals Directory (ACD). The 2D atom types (of the full atomic representation) were assigned and applied as descriptors to encode numerically each compound. There are four main conclusions: First the method performs well, correctly assigning 88% of the compounds in both MDDR and ACD. Improved discrimination was achieved by a more critical selection of training sets. Second, the method gives much better prediction performance than the widely used "Rule of Five", which accepts as many as 74% of the ACD compounds but only 66% of those in MDDR, resulting in a correlation coefficient which is effectively zero, compared to a value of 0.63 for the neural network prediction. Third, based on a standard Tanimoto similarity search the selection of drug-like compounds in the evaluation set is not biased toward compounds similar to those in the training set. Fourth, the trained neural network was applied to evaluate the drug-likeness of 136 GABA uptake inhibitors with impressive results. The implications of applying a neural network to characterize chemical compounds are discussed.
Algorithms↗
A program for the estimation of restriction endonuclease site positions from restriction fragment size and number: an aid for mitochondrial DNA analysis.
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Binding Sites↗