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L J Smith

Publications and source records attributed to L J Smith.

At least 37 records · Page 2Linked to original sources
PubMed2001

Chemical shifts in denatured proteins: resonance assignments for denatured ubiquitin and comparisons with other denatured proteins.

Chemical shift assignment is reported for the protein ubiquitin denatured in 8M urea at pH 2. The variations in 15N chemical shifts of three different proteins (ubiquitin, disulfide reduced, carboxymethylated lysozyme, all-Ala-alpha-lactalbumin), all without disulfides and denatured in 8M urea at pH 2 are compared to 'random coil shifts' of small model peptides (Braun et al., 1994) and to the averaged native chemical shifts taken from the BMRB database. Both parameterizations show a remarkable agreement with the averaged measured 15N chemical shifts in the three denatured proteins. Detailed analysis of these experimental 15N chemical shifts provides an estimate of the influence of nearest neighbors and conformational preferences on the chemical shift and provides a direct means to identify non-random structural preferences in denatured proteins.

Animals↗
PubMed2001

Near-total absence of the cerebellum.

We report five cases of near-total absence of the cerebellum with accompanying pontine hypoplasia. The cerebellar remnant in each case comprised only antero-superior masses, the posterior fossa being otherwise fluid filled. Three of these patients, two teenagers and an infant, presented a fairly consistent clinical and neuroradiological phenotype, and a few similar cases are recorded in the literature. The cerebellar remnant was irregular and asymmetrical, and no ventral pontine prominence was discernible. In at least the older two, cerebellar motor functions were not greatly compromised, and intellectual handicap was of a mild degree. We propose that these cases represent a distinct entity of "near-total absence of the cerebellum with flat ventral pons, and relatively mild clinical affection". All cases have been sporadic, implying that the risk of recurrence within a family may be low. Quite different clinical pictures, of considerably greater severity, are demonstrated in the remaining two cases. One had pontocerebellar hypoplasia type 2, while the other had a complex cerebellar and cerebral malformation.

Adolescent↗
PubMed2001

A refined solution structure of hen lysozyme determined using residual dipolar coupling data.

A high resolution NMR structure of hen lysozyme has been determined using 209 residual 1H-15N dipolar coupling restraints from measurements made in two different dilute liquid crystalline phases (bicelles) in conjunction with a data set of 1632 NOE distance restraints, 110 torsion angle restraints, and 60 hydrogen bond restraints. The ensemble of 50 low-energy calculated structures has an average backbone RMSD of 0.50+/-0.13A to the mean structure and of 1.49+/-0.10A to the crystal structure of hen lysozyme. To assess the importance of the dipolar coupling data in the structure determination, the final structures are compared with an ensemble calculated using an identical protocol but excluding the dipolar coupling restraints. The comparison shows that structures calculated with the dipolar coupling data are more similar to the crystal structure than those calculated without, and have better stereochemical quality. The structures also show improved quality factors when compared with additional dipolar coupling data that were not included in the structure calculations, with orientation-dependent 15N chemical shift changes measured in the bicelle solutions, and with T1/T2 values obtained from 15N relaxation measurements. Analysis of the ensemble of NMR structures and comparisons with crystal structures, 15N relaxation data, and molecular dynamics simulations of hen lysozyme provides a detailed description of the solution structure of this protein and insights into its dynamical behavior.

Animals↗
PubMed2001

Comparative efficacy of inhaled corticosteroids and antileukotriene drugs in asthma.

Asthma is an inflammatory disease of the airways that is best treated by minimising exposure to factors that provoke the inflammation (e.g. allergens) and by administering drugs that reduce the inflammatory response. The cornerstone of asthma treatment is inhaled corticosteroids. Their effectiveness is a result of their potent and broad anti-inflammatory properties. Antileukotriene drugs (leukotriene modifiers) provide an alternative and novel approach to the treatment of asthma. The novelty of these new compounds is that their effectiveness is firmly based on the pathophysiology of asthma, specifically the role played by the cysteinyl leukotrienes. At the same time, the availability of the antileukotriene drugs has stirred debate over when they should be used and how they compare to inhaled corticosteroids. Although the answers are not fully known at this time, the currently available published and presented data are adequate for us to draw some conclusions about their relative effectiveness and role in asthma treatment. The antileukotriene drugs are more effective than placebo, but they are not as effective as inhaled corticosteroids in improving lung function [measured as the forced expiratory volume in 1 second (FEV(1)) or peak expiratory flow rate (PEFR)], reducing beta(2)-agonist use, and decreasing symptom-free days. In contrast, they may have similar beneficial effects on reducing asthma exacerbations and decreasing peripheral blood eosinophil counts. In the absence of knowing a priori the response of an individual patient to treatment with either therapy, the data favour initiating treatment with an inhaled corticosteroid. However, for patients with mild to moderate disease there are a number of circumstances that support using an antileukotriene drug first. A few examples are aspirin intolerance, predominantly exercise-induced symptoms and problems with using an inhaler or the adverse effects of inhaled corticosteroids such as dysphonia and thrush. For patients with more severe disease, inhaled corticosteroids remain the treatment of choice. Antileukotriene drugs should be considered as add-on therapy, especially in view of their possible complementary effects on reducing airway inflammation.

Administration, Inhalation↗
PubMed2001

Response of native and denatured hen lysozyme to high pressure studied by (15)N/(1)H NMR spectroscopy.

High-pressure (15)N/(1)H NMR techniques were used to characterize the conformational fluctuations of hen lysozyme, in its native state and when denatured in 8 M urea, over the pressure range 30--2000 bar. Most (1)H and (15)N signals of native lysozyme show reversible shifts to low field with increasing pressure, the average pressure shifts being 0.069 +/- 0.101 p.p.m. ((1)H) and 0.51 +/- 0.36 p.p.m. ((15)N). The shifts indicate that the hydrogen bonds formed to carbonyl groups or water molecules by the backbone amides are, on average, shortened by approximately 0.02 A as a result of pressure. In native lysozyme, six residues in the beta domain or at the alpha/beta domain interface have anomalously large nonlinear (15)N and (1)H chemical-shift changes. All these residues lie close to water-containing cavities, suggesting that there are conformational changes involving these cavities, or the water molecules within them, at high pressure. The pressure-induced (1)H and (15)N shifts for lysozyme denatured in 8 M urea are much more uniform than those for native lysozyme, with average backbone amide shifts of 0.081 +/- 0.029 p.p.m. ((1)H) and 0.57 +/- 0.14 p.p.m. ((15)N). The results show that overall there are no significant variations in the local conformational properties of denatured lysozyme with pressure, although larger shifts in the vicinity of a persistent hydrophobic cluster indicate that interactions in this part of the sequence may rearrange. NMR diffusion measurements demonstrate that the effective hydrodynamic radius of denatured lysozyme, and hence the global properties of the denatured ensemble, do not change detectably at high pressure.

Animals↗
PubMed2000

A compact monomeric intermediate identified by NMR in the denaturation of dimeric triose phosphate isomerase.

The denaturation of triose phosphate isomerase (TIM) from Saccharomyces cerevisiae by guanidine hydrochlorids at pH 7.2 has been monitored by NMR spectroscopy in conjunction with optical spectroscopy. In the absence of denaturant, the hydrodynamic radius of 29.6(+/-0.25) A and the substantial chemical shift dispersion evident in the NMR spectrum are consistent with the highly structured dimeric native state of the protein. On the addition of 2. 2 M guanidine hydrochloride the effective hydrodynamic radius increases to 51.4(+/-0.43) A, consistent with that anticipated for the polypeptide chain in a highly unstructured random coil state. In 1.1 M guanidine hydrochloride, however, the effective hydrodynamic radius is 24.0(+/-0.25) A, a value substantially decreased relative to that of the native dimeric state but very close to that anticipated for a monomeric species with native-like compaction (23. 5 A). The lack of chemical shift dispersion indicates, however, that few tertiary interactions persist within this species. Far UV CD and intrinsic fluorescence measurements show that this compact intermediate retains significant secondary structure and that on average the fluorophores are partially excluded from solvent. Such a species could be important in the formation of dimeric TIM from its unfolded state.

Circular Dichroism↗
PubMed2000

Identification of residues involved in the interaction of Staphylococcus aureus fibronectin-binding protein with the (4)F1(5)F1 module pair of human fibronectin using heteronuclear NMR spectroscopy.

Many pathogenic Gram-positive bacteria express cell surface proteins that bind to components of the extracellular matrix. This paper describes studies of the interaction between ligand binding repeats (D3 and D1-D4) of a fibronectin-binding protein from Staphylococcus aureus with a module pair ((4)F1(5)F1) from the N-terminal region of fibronectin. When D3 was added to isotope-labeled (4)F1(5)F1, (1)H, (15)N, and (13)C NMR chemical shift changes indicate that binding is primarily via residues in (4)F1, although a few residues in (5)F1 are also affected. Both hydrophobic and electrostatic interactions appear to be involved. The NMR data indicate that part of the D3 repeat converts from a disordered to a more ordered, extended conformation on binding to (4)F1(5)F1. In further NMR experiments, selective reduction of the intensity of D1-D4 resonances was observed on binding to (4)F1(5)F1, consistent with previous suggestions that in each of D1, D2, and D3 repeats, the main fibronectin binding site is in the C-terminal region of the repeat. In D1-D4, these regions also appear to go from a disordered to a more ordered conformation of fibronectin binding. Although the regions of the two proteins which interact had been previously identified, the findings presented here identify, for the first time, the specific residues in both proteins that are likely to be involved in the interaction.

Adhesins, Bacterial↗
PubMed2000

NOF-11: a one-year pediatric randomized double-blind comparison of neoral versus sandimmune in orthotopic liver transplantation.

BACKGROUND: Although cyclosporine (CsA) has been a mainstay in liver transplantation immunosuppression the original formulation [Sandimmune (SIM)] has variable absorption, particularly in children. Neoral is a new formulation of CsA that may have improved biovailability that would be advantageous in children. This study was undertaken to assess the pharmacokinetics (PK) and effects on outcome of Neoral versus Sandimmune (SIM) in primary pediatric liver transplant recipients. METHODS: Thirty-two patients were randomized to receive Neoral (17 patients) or SIM (15 patients) in the early posttransplant period (days 1-7) in a double-blind fashion. Intravenous CsA was instituted immediately posttransplant followed by Neoral or SIM as soon as the patient was tolerating oral fluids (days 1-7). PK were compared after the first dose (1-7 days), 3 weeks, and 6 and 12 months posttransplant. In addition, side effects, effect of age and food on absorption, and rejection episodes were assessed by intent to treat analysis. Notable characteristics of this study include the use of a central laboratory for all sample analyses and the assessment of renal function using radioisotopic evaluation of glomerular filtration rates. RESULTS: At baseline the two groups were comparable. Neoral resulted in higher peak levels of CsA and total drug exposure with comparable time to peak drug levels at days 1-7 and week 3. This trend was maintained at 6 and 12 months. Time on i.v. CsA was reduced in the Neoral group (8.4 vs. 11.1 days) and the weight adjusted daily dose of SIM required to achieve target trough levels was about 2-fold more than Neoral from day 22 onward. In addition, biopsy proven and treated and steroid-resistant rejection episodes were fewer in the Neoral group (6 vs. 12; P=0.01 and 1 vs. 8: P=0.004, respectively). Side effects were comparable in both treatment groups. CONCLUSIONS: Neoral was well tolerated and had greater biovailability than SIM without any increase in the incidence of side effects. In addition fewer episodes of rejection were observed with Neoral versus SIM. We conclude that Neoral is the CsA formulation of choice for use in pediatric liver transplant recipients.

Child↗
PubMed2000

Experience with aggressive therapy in three children with unresectable malignant liver tumors.

BACKGROUND: Children with malignant liver tumors often present with unresectable disease but need not be considered incurable. The advent of effective chemotherapy makes aggressive management feasible, as our experience with three such patients demonstrates. Procedure and Results One child with an unresectable undifferentiated sarcoma of the liver and two others with unresectable primary hepatoblastoma and lung metastases were treated with initial chemotherapy, followed by aggressive surgical management. Treatment with chemotherapy followed by hepatectomy and liver transplantation (cadaveric or live donor) in two children has resulted in disease-free survivals of 79 and 38 months. The third patient is alive and well 24 months following chemotherapy and aggressive resection of the primary and 12 metastatic lesions. CONCLUSIONS: Initial chemotherapy for unresectable liver tumors with or without metastases is supported by the review of the literature. Consideration of orthotopic liver transplantation (OLT) from cadaveric or living related donor is warranted when the malignancy is demonstrably chemosensitive, independent of initial staging. Aggressive resection of primary and metastatic disease may be called for in selected cases.

Child↗
PubMed2000

Understanding protein folding via free-energy surfaces from theory and experiment.

The ability of protein molecules to fold into their highly structured functional states is one of the most remarkable evolutionary achievements of biology. In recent years, our understanding of the way in which this complex self-assembly process takes place has increased dramatically. Much of the reason for this advance has been the development of energy surfaces (landscapes), which allow the folding reaction to be described and visualized in a meaningful manner. Analysis of these surfaces, derived from the constructive interplay between theory and experiment, has led to the development of a unified mechanism for folding and a recognition of the underlying factors that control the rates and products of the folding process.

Animals↗
PubMed2000

Encephalocraniocutaneous lipomatosis (Fishman syndrome): a rare neurocutaneous syndrome.

Encephalocraniocutaneous lipomatosis (ECCL) is a rare congenital neurocutaneous syndrome comprising unilateral cranial lipomas, lipodermoids of the eye and brain abnormalities. A 3-year-old boy who presented at birth with a scalp lipoma and an ipsilateral epibulbar lipodermoid is described. Infantile spasms developed at 9 months of age and cerebral imaging showed thickened and calcified cortex at the right occiput and hemiatrophy of the right hemisphere. These features were consistent with ECCL. Most children with ECCL have significant developmental delay, but we have found that control of seizures was associated with a significant improvement in developmental outcome.

Brain↗
PubMed2000

Syntelencephaly presenting with spastic diplegia.

Two children with syntelencephaly presented with the predominant clinical picture of spastic diplegia. Brain scanning showed deficient formation of the interhemispheric fissure with fusion of occipital and parietal lobes, and an infolding and protrusion of occipital cortex into the telencephalic ventricle. The fusion extended further posteriorly than the cases of "middle interhemispheric fusion" thus far reported. One 7-year old has poor language development, while the other is regarded, at her present age of 3 years, as being cognitively within the normal range.

Agenesis of Corpus Callosum↗
PubMed2000

Initial denaturing conditions influence the slow folding phase of acylphosphatase associated with proline isomerization.

The folding kinetics of human common-type acylphosphatase (cAcP) from its urea- and TFE-denatured states have been determined by stopped-flow fluorescence techniques. The refolding reaction from the highly unfolded state formed in urea is characterized by double exponential behavior that includes a slow phase associated with isomerism of the Gly53-Pro54 peptide bond. However, this slow phase is absent when refolding is initiated by dilution of the highly a-helical denatured state formed in the presence of 40% trifluoroethanol (TFE). NMR studies of a peptide fragment corresponding to residues Gly53-Gly69 of cAcP indicate that only the native-like trans isomer of the Gly-Pro peptide bond is significantly populated in the presence of TFE, whereas both the cis and trans isomers are found in an approximately 1:9 ratio for the peptide bond in aqueous solution. Molecular modeling studies in conjunction with NMR experiments suggest that the trans isomer of the Gly53-Pro54 peptide bond is stabilized in TFE by the formation of a nonnative-like hydrogen bond between the CO group of Gly53 and the NH group of Lys57. These results therefore reveal that a specific nonnative interaction in the denatured state can increase significantly the overall efficiency of refolding.

Acid Anhydride Hydrolases↗
PubMed2000

Tobramycin solution for inhalation reduces sputum Pseudomonas aeruginosa density in bronchiectasis.

We conducted a placebo-controlled, double-blind, randomized study to evaluate the microbiological efficacy and safety of inhaled tobramycin for treatment of patients with bronchiectasis and Pseudomonas aeruginosa. Patients were randomly assigned to receive either tobramycin solution for inhalation (TSI) (n = 37) or placebo (n = 37), which was self-administered twice daily for 4 wk and followed by 2-wk off-drug. At Week 4, the TSI group had a mean decrease in P. aeruginosa density of 4.54 log(10) colony-forming units (cfu)/g sputum compared with no change in the placebo group (p < 0.01). At Week 6, P. aeruginosa was eradicated in 35% of TSI patients but was detected in all placebo patients. Investigators indicated that 62% of TSI patients showed an improved medical condition compared with 38% of placebo patients (odds ratio = 2.7, 95% confidence interval [CI] 1.1 to 6.9). Tobramycin-resistant P. aeruginosa strains developed in 11% of TSI patients and 3% of placebo patients (p = 0.36). The mean percent change in FEV(1) percent predicted from Week 0 to Week 4 was similar for the TSI and placebo groups (p = 0.41). More TSI-treated patients than placebo patients reported increased cough, dyspnea, wheezing, and noncardiac chest pain, but the symptoms did not limit therapy. Additional study is warranted to further evaluate TSI in bronchiectasis patients.

Administration, Inhalation↗
PubMed1999

Hydrodynamic radii of native and denatured proteins measured by pulse field gradient NMR techniques.

Pulse field gradient NMR methods have been used to determine the effective hydrodynamic radii of a range of native and nonnative protein conformations. From these experimental data, empirical relationships between the measured hydrodynamic radius (R(h)) and the number of residues in the polypeptide chain (N) have been established; for native folded proteins R(h) = 4.75N (0.29)A and for highly denatured states R(h) = 2.21N (0.57)A. Predictions from these equations agree well with experimental data from dynamic light scattering and small-angle X-ray or neutron scattering studies reported in the literature for proteins ranging in size from 58 to 760 amino acid residues. The predicted values of the hydrodynamic radii provide a framework that can be used to analyze the conformational properties of a range of nonnative states of proteins. Several examples are given here to illustrate this approach including data for partially structured molten globule states and for proteins that are unfolded but biologically active under physiological conditions. These reveal evidence for significant coupling between local and global features of the conformational ensembles adopted in such states. In particular, the effective dimensions of the polypeptide chain are found to depend significantly on the level of persistence of regions of secondary structure or features such as hydrophobic clusters within a conformational ensemble.

Adhesins, Bacterial↗
PubMed1999

Molecular dynamics simulations of human alpha-lactalbumin: changes to the structural and dynamical properties of the protein at low pH.

Two 700-ps molecular dynamics simulations of human alpha-lactalbumin have been compared. Both were initiated from an X-ray structure determined at pH 6.5. One simulation was designed to represent native conditions and the other the protein in solution at pH 2.0 without a bound calcium ion. The low pH conditions were modelled by protonating the aspartate, glutamate, and histidine side chains and the protein C-terminus. Significant changes were observed for the C-terminal region of the sequence in the simulation at low pH. Most notably an alpha-helix, helix D, and the C-terminal 3(10) helix were substantially disrupted relative to the simulation at high pH. These perturbations to the native fold are similar to those observed in an X-ray structure of alpha-lactalbumin at pH 4.2. In addition, larger fluctuations about side chain torsion angles were observed in the low pH simulation than in that corresponding to the higher pH. These structural and dynamical changes might be representative of the early stages of the transition to the molten-globule state of the protein known to be formed under low pH conditions in solution.

Humans↗
PubMed1999

Side-chain conformations in an unfolded protein: chi1 distributions in denatured hen lysozyme determined by heteronuclear 13C, 15N NMR spectroscopy.

Using a 13C and 15N-labelled sample, multi-dimensional heteronuclear NMR techniques have been carried out to characterise hen lysozyme denatured in 8 M urea at pH 2.0. The measurement of 3J(C',Cgamma) and 3J(N,Cgamma) coupling constants has enabled side-chain chi1 torsion angle populations to be probed in the denatured polypeptide chain. Analysis of the coupling constant data has allowed the relative populations of the three staggered rotamers about chi1 to be defined for 51 residues. The amino acids can broadly be divided into five classes that show differing side-chain conformational preferences in the denatured state. These range from a strong preference for the -60 degrees chi1 rotamer for methionine and leucine (74-79 % population) to a favouring of the +60 degrees chi1 rotamer for threonine (67 % population). The differences in behaviour reflect the steric and electrostatic characteristics of the side-chains concerned. A close agreement is seen between the chi1 populations calculated from the experimental coupling constant data and predictions from the statistical model for a random coil that uses the chi1 torsion angle distributions in a data base of native protein structures. Short-range interactions therefore dominate in determining the local conformational properties of side-chains in a denatured protein. Deviations are, however, observed for many of the aromatic residues involved in hydrophobic clusters within the denatured protein. For these residues the effects of additional non-local interactions in the clusters presumably play a major role in determining the chi1 preferences.

Animals↗