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D H Williams

Publications and source records attributed to D H Williams.

At least 37 records · Page 2Linked to original sources
PubMed2000

Structural characterization of a mutant peptide derived from ubiquitin: implications for protein folding.

The formation of the N-terminal beta-hairpin of ubiquitin is thought to be an early event in the folding of this small protein. Previously, we have shown that a peptide corresponding to residues 1-17 of ubiquitin folds autonomously and is likely to have a native-like hairpin register. To investigate the causes of the stability of this fold, we have made mutations in the amino acids at the apex of the turn. We find that in a peptide where Thr9 is replaced by Asp, U(1-17)T9D, the native conformation is stabilized with respect to the wild-type sequence, so much so that we are able to characterize the structure of the mutant peptide fully by NMR spectroscopy. The data indicate that U(1-17)T9D peptide does indeed form a hairpin with a native-like register and a type I turn with a G1 beta-bulge, as in the full-length protein. The reason for the greater stability of the U(1-17)T9D mutant remains uncertain, but there are nuclear Overhauser effects between the side chains of Asp9 and Lys 11, which may indicate that a charge-charge interaction between these residues is responsible.

Amino Acid Sequence↗
PubMed2000

Spinal dysraphism at MR urography: initial experience.

PURPOSE: To prospectively evaluate the role of magnetic resonance (MR) urography in the radiologic assessment of patients with spinal dysraphism. MATERIALS AND METHODS: Fourteen patients with spinal dysraphism were referred for MR urography with half-Fourier rapid acquisition with relaxation enhancement (RARE) (repetition time msec/echo time msec = 11.9/95) and RARE (2,800/1,100) sequences on a 1.5-T MR machine. Six patients did not tolerate MR urography owing to claustrophobia (n = 4) or flexion deformities (n = 2), giving a final success rate of 57% (eight patients). Two patients had a single kidney (one after nephrectomy, one with a crossed-fused ectopic kidney). Images were jointly assessed by two radiologists and compared with excretory urographic studies. The signal intensity ratio and contrast-to-noise ratio were also calculated. RESULTS: Visualization of the kidneys, pelvicaliceal system, and ureters was better with half-Fourier RARE than with RARE imaging, whereas visualization of the bladder was comparable with both sequences. The mean signal intensity ratios for half-Fourier RARE and RARE sequences, respectively, were 16.2 +/- 3.65 and 19.2 +/- 3.65 (P =. 58, factorial analysis of variance model), whereas the mean contrast-to-noise ratios were 55.4 +/- 5.16 and 47.8 +/- 5.16 (P =. 34). Cortical scarring was depicted more clearly at MR urography than at excretory urography, whereas a renal calculus seen at excretory urography was not detected at MR urography. CONCLUSION: MR urography was feasible in 57% of patients with spinal dysraphism and is a safe, accurate method of evaluating the upper urinary tract.

Adolescent↗
PubMed1999

Kinetic analysis of antibody-antigen interactions at a supported lipid monolayer.

Modified phospholipids possessing carboxyl head groups synthesized from phosphatidylethanolamine were incorporated into supported lipid monolayers on top of a thin gold film. A monoclonal antibody was chemically coupled to the modified lipids in these monolayers and the kinetics of antigen binding were determined by surface plasmon resonance. The binding could be analyzed using a conventional 1:1 binding algorithm and the derived kinetic and affinity constants were almost identical to those reported for the same interaction on a dextran hydrogel-based sensor chip. When an antigen was chemically coupled to a modified lipid monolayer, the binding of a monoclonal antibody to this surface was biphasic. A two-step algorithm describing the formation of a 1:2 antibody:antigen complex was developed which accurately described the data and enabled differentiation of the two binding steps. The binding was assayed varying both the concentration of antibody in solution and the density of antigen on the surface. The affinities determined by Scatchard analysis of equilibrium binding levels were similar to those values obtained from an ELISA.

Animals↗
PubMed1999

Structure of the protein tyrosine kinase domain of C-terminal Src kinase (CSK) in complex with staurosporine.

The crystal structure of the kinase domain of C-terminal Src kinase (CSK) has been determined by molecular replacement, co-complexed with the protein kinase inhibitor staurosporine (crystals belong to the space group P21212 with a=44.5 A, b=120.6 A, c=48.3 A). The final model of CSK has been refined to an R-factor of 19.9 % (Rfree=28.7 %) at 2.4 A resolution. The structure consists of a small, N-terminal lobe made up mostly of a beta-sheet, and a larger C-terminal lobe made up mostly of alpha-helices. The structure reveals atomic details of interactions with staurosporine, which binds in a deep cleft between the lobes. The polypeptide chain fold of CSK is most similar to c-Src, Hck and fibroblast growth factor receptor 1 kinase (FGFR1K) and most dissimilar to insulin receptor kinase (IRK). Interactions between the N and C-terminal lobe are mediated by the bound staurosporine molecule and by hydrogen bonds. In addition, there are several water molecules forming lobe-bridging hydrogen bonds, which may be important for maintaining the catalytic integrity of the kinase. Furthermore, the conserved Lys328 and Glu267 residues utilise water in the formation of a molecular pivot which is essential in allowing relative movement of the N and C-terminal lobes. An analysis of the residues around the ATP-binding site reveals structural differences with other protein tyrosine kinases. Most notable of these are different orientations of the conserved residues Asp332 and Phe333, suggesting that inhibitor binding proceeds via an induced fit. These structural observations have implications for understanding protein tyrosine kinase catalytic mechanisms and for the design of ATP-mimicking inhibitors of protein kinases.

Amino Acid Sequence↗
PubMed1999

Binding of glycopeptide antibiotics to a model of a vancomycin-resistant bacterium.

BACKGROUND: The vancomycin group of glycopeptide antibiotics is active against a wide range of gram-positive bacteria. The increasing resistance to vancomycin is the result of a change of an amide linkage (D-Ala-D-Ala) to an ester linkage (D-Ala-D-Lactate) in the bacterial cell-wall precursors. RESULTS: We have used a peptide terminating in the sequence -Lys-D-Ala-D-Lactate linked by its amino terminus to a docosanoyl (C22) acyl chain and anchored in a supported lipid monolayer to mimic the surface of vancomycin-resistant enterococci. Surface plasmon resonance analysis was then used to investigate the binding of glycopeptide group antibiotics to this surface. Vancomycin, which dimerises weakly, bound with low affinity, whereas strongly dimerising antibiotics, such as chloroeremomycin, bound with higher affinities. Antibiotics that have attached hydrophobic groups, such as teicoplanin and biphenylchloroeremomycin (LY307599), bound to the lipid monolayer. This resulted in an enhanced affinity for the lipid-anchored peptide at the surface relative to affinities for an analogous non-anchored peptide in solution. CONCLUSIONS: We have shown that the affinities of glycopeptide antibiotics for a model of the surface of a vancomycin-resistant bacterium are enhanced relative to affinities determined in free solution. We have also shown that antibiotics that have membrane anchors bind tightly to the model surface and that this feature is an important determinant of the ability of an antibiotic to kill vancomycin-resistant enterococci.

Anti-Bacterial Agents↗
PubMed1999

Autonomous folding of a peptide corresponding to the N-terminal beta-hairpin from ubiquitin.

The N-terminal 17 residues of ubiquitin have been shown by 1H NMR to fold autonomously into a beta-hairpin structure in aqueous solution. This structure has a specific, native-like register, though side-chain contacts differ in detail from those observed in the intact protein. An autonomously folding hairpin has previously been identified in the case of streptococcal protein G, which is structurally homologous with ubiquitin, but remarkably, the two are not in topologically equivalent positions in the fold. This suggests that the organization of folding may be quite different for proteins sharing similar tertiary structures. Two smaller peptides have also been studied, corresponding to the isolated arms of the N-terminal hairpin of ubiquitin, and significant differences from simple random coil predictions observed in the spectra of these subfragments, suggestive of significant limitation of the backbone conformational space sampled, presumably as a consequence of the strongly beta-structure favoring composition of the sequences. This illustrates the ability of local sequence elements to express a propensity for beta-structure even in the absence of actual sheet formation. Attempts were made to estimate the population of the folded state of the hairpin, in terms of a simple two-state folding model. Using published "random coil" values to model the unfolded state, and values derived from native ubiquitin for the putative unique, folded state, it was found that the apparent population varied widely for different residues and with different NMR parameters. Use of the spectra of the subfragment peptides to provide a more realistic model of the unfolded state led to better agreement in the estimates that could be obtained from chemical shift and coupling constant measurements, while making it clear that some other approaches to population estimation could not give meaningful results, because of the tendency to populate the beta-region of conformational space even in the absence of the hairpin structure.

Magnetic Resonance Spectroscopy↗
PubMed1999

Effect of duration of dominance of the ovulatory follicle on onset of estrus and fertility in heifers.

In cattle, prolonged progestogen treatments following luteolysis result in persistent dominant follicles (DF) that are associated with precise onset of estrus but marked reductions in pregnancy rate (PR). The aim was to determine whether increasing duration of dominance of the ovulatory follicle in heifers affected 1) precision of onset of estrus and 2) the timing and nature of the decline in PR. In Exp. 1, duration of dominance of the ovulatory follicle was controlled by causing corpus luteum (CL) regression at emergence of the second follicle wave (mean duration of dominance of 2.1+/-.3 d, Dm2, n = 11) or first day of dominance of the second DF of the cycle; the latter was combined with insertion of a 3-mg norgestomet ear implant for 2 to 10 d to maintain the second DF for 4 (Dm4, n = 32), 6 (Dm6, n = 19), 8 (Dm8, n = 49), 10 (Dm10, n = 28), or 12 d (Dm12, n = 20). Heifers detected in estrus were inseminated approximately 12 h later with frozen-thawed semen. Durations of dominance of the ovulatory follicle of up to 8 d did not affect (P>.05) PR (Dm2 8/9, Dm4 19/28, Dm6 14/18, and Dm8 34/48 heifers pregnant), but PR in Dm10 heifers (12/23 heifers pregnant) was reduced (P = .05) compared with Dm2 heifers; PR in Dm12 heifers (2/17 pregnant) was less compared with all other treatments (P<.01). Fitting a logistic regression model to the pooled PR data to examine the trend in PR showed that extending the duration of dominance from 2 to 9 d and from 10 to 12 d resulted in a predicted decline in PR of 10 to 25% and a further decline of 35 to 75%, respectively. Onset of estrus was delayed in heifers assigned to Dm4 treatment relative to all other treatments (P<.001); it was less variable than that for heifers on Dm6, Dm8, and Dm10 treatments (P<.1). In Exp. 2, heifers received a PGF2alpha analogue and a norgestomet implant on d 12 of the cycle for 3 or 7 d to give approximate durations of dominance of the preovulatory follicle of 2 to 4 d (Dm2-4, n = 29) or 6 to 8 d (Dm6-8, n = 24), respectively. The PR did not differ (P>.05) between heifers on Dm2-4 (22/29) and Dm6-8 (15/24) treatments, but the interval to onset of estrus was delayed (P<.05) by 7 h in the Dm2-4 heifers. In conclusion, restricting the duration of dominance of the preovulatory follicle to < or =4 d at estrus, results in a precise onset of estrus and a high PR following a single AI at a detected estrus.

Animals↗
PubMed1998

Human MAFA has alternatively spliced variants.

Human mast cell function-associated antigen (MAFA) cDNA has been cloned. This molecule is similar to the rat form having an intracellular domain containing a putative immunoreceptor tyrosine inhibition motif and an extracellular C type lectin-like domain. However, in contrast to rat MAFA, the amino acid sequence suggests the presence of two additional extracellular N-linked glycosylation sites. In addition, alternative mRNA transcripts are observed that differ substantially from those found in the rat.

Alternative Splicing↗
PubMed1998

Surface plasmon resonance analysis at a supported lipid monolayer.

Methods for the formation of supported lipid monolayers on top of a hydrophobic self assembled monolayer in a surface plasmon resonance instrument are described. Small unilamellar vesicles absorb spontaneously to the surface of the hydrophobic self-assembled monolayer to form a surface which resembles the surface of a cellular membrane. Lipophilic ligands, such as small acylated peptides or glycosylphosphatidylinositol-anchored proteins, were inserted into the absorbed lipid and binding of analytes to these ligands was analysed by surface plasmon resonance. Conditions for the formation of lipid monolayers have been optimised with respect to lipid type, chemical and buffer compatibility, ligand stability and reproducibility.

Anti-Bacterial Agents↗
PubMed1998

Bacillus thuringiensis Cry1Ac toxin interaction with Manduca sexta aminopeptidase N in a model membrane environment.

The Bacillus thuringiensis Cry1Ac delta-endotoxin was shown to bind in a biphasic manner to Manduca sexta aminopeptidase N (APN) present in a novel model membrane. Surface plasmon resonance analysis allowed the quantification of toxin binding to M. sexta APN in a supported lipid monolayer. The initial binding was rapid and reversible, with an affinity constant of 110 nM. The second phase was slower and resulted in an overall affinity constant of 3.0 nM. Reagents used to disrupt protein-protein interactions did not dissociate the toxin after high-affinity binding was attained. The initial association between Cry1Ac and APN was inhibited by the sugar GalNAc, but the higher-affinity state was resistant to GalNAc-induced dissociation. The results suggest that after binding to M. sexta APN, the Cry1Ac toxin undergoes a rate-limiting step leading to a high-affinity state. A site-directed Cry1Ac mutant, N135Q, exhibited a similar initial binding affinity for APN but did not show the second slower phase. This inability to form an irreversible association with the APN-lipid monolayer helps explain the lack of toxicity of this protein towards M. sexta larvae and its deficient membrane-permeabilizing activity on M. sexta midgut brush border membrane vesicles.

Animals↗
PubMed1998

Ro 09-2210 exhibits potent anti-proliferative effects on activated T cells by selectively blocking MKK activity.

By using high throughput screening of microbial broths, we have identified a compound, designated Ro 09-2210, which is able to block anti-CD3 induced peripheral blood T cell activation with an IC50 = 40 nM. Ro 09-2210 was also able to block antigen-induced IL-2 secretion with an IC50 = 30 nM, but was considerably less potent at blocking Ca2+ flux stimulated by anti-CD3 treatment. To determine the mechanism of action of Ro 09-2210, we set up a transient expression system in Jurkat T cells using a variety of reporter gene constructs and showed effective inhibition of phorbol ester/ionomycin-induced NF-AT activation and anti-CD3 induced NF-AT with IC50 = 7.7 and 10 nM, respectively. Ro 09-2210 was also able to inhibit phorbol ester/ionomycin-induced activation of AP1 with IC50 = <10 nM. We further showed that Ro 09-2210 was unable to inhibit c-jun induced expression of AP1-dependent reporter constructs (IC50 > 500 nM), but was able to potently inhibit ras-induced AP1 activation (IC50 = 20 nM). This suggested that Ro 09-2210 was inhibiting an activator of AP-1 which was upstream of c-jun and downstream of ras signaling. To investigate further, we then purified a number of different kinases, including PKC, PhK, ZAP-70, ERK, and MEK 1 (a MKK), and showed that Ro 09-2210 was a selective inhibitor of MEK1 in vitro (IC50 = 59 nM).

DNA-Binding Proteins↗
PubMed1998

The influence of selected herd factors and a badger-intervention tuberculosis-control programme on the risk of a herd-level trade restriction to a bovine population in Ireland.

The risk of a trade restriction to cattle herds between 1990-1994, as a result of failing the tuberculin skin test or disclosure of visible lesions in the abattoir, was compared in two areas in East Offaly, Ireland. Cattle herds from the Project area where badgers were systematically removed over a 7-year period (1989-1995) were compared to herds from a Control area where no wildlife-intervention programme had taken place. A Cox proportional hazard model indicated, for example, that the risk of a herd-level trade restriction to herds with 30 animals in the Control area was 1.4 times the risk to similar-sized herds in the Project area. The study provides evidence that a badger-control programme is effective in reducing the risk of a trade restriction to cattle herds as the badger represents an important reservoir of Mycobacterium bovis infection for cattle.

Animals↗
PubMed1998

An analysis of the origins of a cooperative binding energy of dimerization.

The cooperativity between binding of cell wall precursor analogs (ligands) to and antibiotic dimerization of the clinically important vancomycin group antibiotics was investigated by nuclear magnetic resonance. When dimerization was weak in the absence of a ligand, the increase in the dimerization constant in the presence of a ligand derived largely from changes associated with tightening of the dimer interface. When dimerization was strong in the absence of a ligand, the increase in the dimerization constant in the presence of a ligand derived largely from changes associated with tightening of the ligand-antibiotic interface. These results illustrate how, when a protein has a loose structure, the binding energy of another molecule to the protein can derive in part from changes occurring within the protein.

Anti-Bacterial Agents↗
PubMed1998

The effect of a badger removal programme on the incidence of tuberculosis in an Irish cattle population.

The risk of a confirmed tuberculous herd restriction was examined using a logistic model for herds involved in the East Offaly Badger Research Project, Ireland, from 1988-1995. Cattle herds present in the badger-removal area had a significantly lower proportion of new confirmed tuberculous herd restrictions compared with cattle from an area where no systematic badger removal was attempted.

Animals↗
PubMed1998

Modelling the prevalence of Echinococcus and Taenia species in small ruminants of different ages in northern Jordan.

A base-line survey of Echinococcus granulosus, Taenia hydatigena and T ovis were undertaken in order to investigate the transmission dynamics of these parasites in northern Jordan. Intensity of E. granulosus infection, in sheep, increased with age in a linear fashion whilst the asymptotic prevalence was one. This implied that E. granulosus is in an endemic steady state with no evidence of protective immunity in the intermediate host. The mean number of cysts increased by 1.66 per year with approximately 0.320 infections per year, each infection consisting of 598 eggs to produce 5.2 cysts. The basic reproduction ratio (R0) was estimated to be 1.5-1.8. A similar pattern was suggested with E. granulosus in goats but the infection pressure appeared to be lower with only 0.128 cysts per year. Although infection in goats appeared to be endemic there was some evidence of departure from the model which might indicate that the model needs adjusting for this species. In the case of T. hydatigena the host age-intensity helminth distribution indicated that this parasite was hyperendemic in both sheep and goats, implying regulation by intermediate host immunity. Consequently, R0 was determined from asymptotic prevalence curves for T hydatigena and was calculated to be 4.0 and 3.1 in sheep and goats, respectively. The lower R0 in goats, together with the higher asymptotic age-intensity and age-prevalences, indicates that goats acquire immunity more slowly to T hydatigena in comparison to sheep. Taenia ovis was not detected in any animals.

Animals↗
PubMed1998

Sequencing and analysis of genes involved in the biosynthesis of a vancomycin group antibiotic.

BACKGROUND: The emergence of resistance to vancomycin, the drug of choice against methicillin-resistant Staphylococcus aureus, in enterococci has increased the need for new antibiotics. As chemical modification of the antibiotic structure is not trivial, we have initiated studies towards enzymatic modification by sequencing the DNA coding for the biosynthesis of chloroeremomycin (also known as A82846B and LY264826). RESULTS: Analysis of 72 kilobases of genomic DNA from Amycolatopsis orientalis, the organism that produces chloroeremomycin, revealed the presence of 39 putative genes, including those coding for the biosynthesis of the antibiotic. Translation and subsequent comparison with known proteins in public databases identified enzymes responsible for the biosynthesis of the heptapeptide backbone and 4-epi-vancosamine, as well as those for chlorination and oxidation reactions involved in the biosynthesis of chloroeremomycin. CONCLUSIONS: The genes responsible for the biosynthesis of chloroeremomycin have been identified, and selective expression of these genes could lead to the synthesis of new potent glycopeptide antibiotics.

Actinobacteria↗
PubMed1998

19F NMR in the measurement of binding affinities of chloroeremomycin to model bacterial cell-wall surfaces that mimic VanA and VanB resistance.

BACKGROUND: The emergence of bacteria that are resistant to vancomycin, the drug of choice against methicillin-resistant Staphylococcus aureus, has made the study of the binding characteristics of glycopeptides to biologically relevant depsipeptides important. These depsipeptides, terminating in D-alanyl-D-lactate, mimic the cell-wall precursors of resistant bacteria. RESULTS: The use of 19F-labelled ligands in the study of the therapeutically important vancomycin series of antibiotics is demonstrated. The substantial simplification of spectra that occurs when such labelled ligands are employed is used in the measurement of binding affinities of depsipeptides to chloroeremomycin (CE). Large enhancements of binding affinities are found at a model bacterial cell-wall surface (constituted from depsipeptides that are anchored into vesicles) relative to those measured in free solution. CONCLUSIONS: Surface-enhanced binding, previously shown for strongly dimerizing glycopeptide antibiotics to normal -D-alanyl-D-alanine-terminating cell-wall precursors, is now demonstrated for CE to the surface of models of VanA- and VanB-resistant bacteria. The effect of depsipeptide chain length is shown to be critically important in producing and maximizing this enhancement.

Anti-Bacterial Agents↗