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Biomedical subjects

J Kroon

Publications and source records attributed to J Kroon.

At least 19 recordsLinked to original sources
PubMed2001

Conformational comparison of five follicular fluid meiosis-activating sterol-related active and inactive compounds.

The crystal structures of five follicular fluid meiosis-activating sterol-related Delta(8,14)-sterol compounds are presented. These are 4,4-dimethyl-23-phenyl-24-nor-5alpha-chola-8,14-dien-3beta-ol, C(31)H(44)O, 4,4-dimethyl-22-phenyl-23,24-dinor-5alpha-chola-8,14-dien-3beta-ol, C(30)H(42)O, (20R)-4,4-dimethyl-22-oxa-5alpha,20-cholesta-8,14,24-trien-3beta-ol, C(28)H(44)O(2), 4,4-dimethyl-23-phenyl-22-oxa-24-nor-5alpha-chola-8,14-dien-3beta-ol-water (4/1), 4C(30)H(42)O(2).H(2)O, and 4,4-dimethyl-5alpha-cholesta-8,14-dien-3-one, C(29)H(46)O. Two of the derivatives are inactive and three are active as agonists. Preliminary structure-activity relationship studies showed that the positions of the double bonds in the skeleton and the structures of the side chains are important determinants for activity. The conformations of the skeletons were compared with double-bond isomers retrieved from the Cambridge Structural Database [Allen & Kennard (1993). Chem. Des. Autom. News, 8, 1, 31-37]; no significant differences were found. Thus, conformational changes induced by the double bonds are not discriminative with respect to the activity of the compounds. Comparisons of the side-chain conformations of active and inactive structures revealed that the crystal structures were not conclusive as far as correlation of conformation and activity of the side chains were concerned.

Crystallography, X-Ray↗
PubMed2001

Crystal structure of the outer membrane protease OmpT from Escherichia coli suggests a novel catalytic site.

OmpT from Escherichia coli belongs to a family of highly homologous outer membrane proteases, known as omptins, which are implicated in the virulence of several pathogenic Gram-negative bacteria. Here we present the crystal structure of OmpT, which shows a 10-stranded antiparallel beta-barrel that protrudes far from the lipid bilayer into the extracellular space. We identified a putative binding site for lipopolysaccharide, a molecule that is essential for OmpT activity. The proteolytic site is located in a groove at the extracellular top of the vase-shaped beta-barrel. Based on the constellation of active site residues, we propose a novel proteolytic mechanism, involving a His-Asp dyad and an Asp-Asp couple that activate a putative nucleophilic water molecule. The active site is fully conserved within the omptin family. Therefore, the structure described here provides a sound basis for the design of drugs against omptin-mediated bacterial pathogenesis. Coordinates are in the Protein Data Bank (accession No. 1I78)

Amino Acid Sequence↗
PubMed2001

SuperStar: comparison of CSD and PDB-based interaction fields as a basis for the prediction of protein-ligand interactions.

SuperStar is an empirical method for identifying interaction sites in proteins, based entirely on the experimental information about non-bonded interactions, present in the IsoStar database. The interaction information in IsoStar is contained in scatterplots, which show the distribution of a chosen probe around structure fragments. SuperStar breaks a template molecule (e.g. a protein binding site) into structural fragments which correspond to those in the scatterplots. The scatterplots are then superimposed on the corresponding parts of the template and converted into a composite propensity map. The original version of SuperStar was based entirely on scatterplots from the CSD. Here, scatterplots based on protein-ligand interactions are implemented in SuperStar, and validated on a test set of 122 X-ray structures of protein-ligand complexes. In this validation, propensity maps are compared with the experimentally observed positions of ligand atoms of comparable types. Although non-bonded interaction geometries in small molecule structures are similar to those found in protein-ligand complexes, their relative frequencies of occurrence are different. Polar interactions are more common in the first class of structures, while interactions between hydrophobic groups are more common in protein crystals. In general, PDB and CSD-based SuperStar maps appear equally successful in the prediction of protein-ligand interactions. PDB-based maps are more suitable to identify hydrophobic pockets, and inherently take into account the experimental uncertainties of protein atomic positions. If the protonation state of a histidine, aspartate or glutamate protein side-chain is known, specific CSD-based maps for that protonation state are preferred over PDB-based maps which represent an ensemble of protonation states.

Databases, Factual↗
PubMed2001

The structure of leech anti-platelet protein, an inhibitor of haemostasis.

Leech anti-platelet protein (LAPP) from the leech Haementeria officinalis is a collagen-binding protein that inhibits the collagen-mediated adhesion of blood platelets. The crystal structure of recombinant LAPP has been determined using single isomorphous replacement with anomalous scattering combined with solvent flattening and threefold molecular averaging. The model of LAPP has been refined to 2.2 A resolution (R factor 21.5%; free R factor 24.0%). LAPP contains an 89-residue C-terminal domain consisting of a central six-stranded antiparallel beta-sheet flanked on one side by an alpha-helix and on the other side by two extended loops with little secondary structure. A 36-residue N-terminal region is not visible in the electron-density map. This region is rich in glycine and lacks hydrophobic residues. It probably does not have a compact globular fold, but instead has an extended conformation and is flexible. The crystal packing suggests that LAPP may form tightly interacting dimers. The fold of the C-terminal domain of LAPP closely resembles that of the N-domain of hepatocyte growth factor (HGF), which classifies LAPP as a PAN domain. However, no significant sequence homology exists between LAPP and other PAN domains. Common structural features between LAPP and the HGF N-domain include two disulfide bonds that link the alpha-helix to the central region of the protein and five residues with a conserved hydrophobic nature that are located in the core of the domain. These conserved structural features may be an important determinant of the PAN-domain type of fold.

Amino Acid Sequence↗
PubMed2001

Biophysical characterization of the influence of salt on tetrameric SecB.

SecB is a tetrameric chaperone, with a monomeric molecular mass of 17 kDa, that is involved in protein translocation in Escherichia coli. It has been hypothesized that SecB undergoes a conformational change as a function of the salt concentration. To gain more insight into the salt-dependent behavior of SecB, we studied the protein in solution by dynamic light scattering, size exclusion chromatography, analytical ultracentrifugation, and small angle neutron scattering. The results clearly demonstrate the large influence of the salt concentration on the behavior of SecB. At high salt concentration, SecB is a non-spherical protein with a radius of gyration of 3.4 nm. At low salt concentration the hydrodynamic radius of the protein is apparently decreased, whereas the ratio of the frictional coefficients is increased. The protein solution behaves in a non-ideal way at low salt concentrations, as was shown by the analytical ultracentrifugation data and a pronounced interparticle effect observed by small angle neutron scattering. A possible explanation is a change in surface charge distribution dependent on the salt concentration in the solvent. We summarize our data in a model for the salt-dependent conformation of tetrameric SecB.

Bacterial Proteins↗
PubMed2001

Relation between the molecular electrostatic potential and activity of some FF-MAS related sterol compounds.

Follicular Fluid-Meiosis Activating Sterol (FF-MAS) is a compound important for maturation of gametes in mammals. Therefore, it may serve as a lead compound for a novel method of contraception. We studied the Molecular Electrostatic Potential of a series of active and inactive analogues of FF-MAS. We find that double bond configurations required for activity result in a local negative electrostatic potential which is larger as well as more dense compared to those of inactive molecules. We therefore hypothesize that the interaction energy of the double bond system of the MAS compounds with its receptor substantially contributes to the overall interaction energy. This notion is supported by interaction studies of the electrostatic potential originating from the double bonds in crystal structures of cholesterol and four MAS-derived Delta(8,14) structures synthesized and crystallized by us. In addition, we were able to derive a pharmacophore model that relates the local average ESP and its distance to the 3beta-OH oxygen atom to the activity of the molecules.

Animals↗
PubMed2001

Solvent behaviour in flash-cooled protein crystals at cryogenic temperatures.

The solvent behaviour of flash-cooled protein crystals was studied in the range 100--180 K by X-ray diffraction. If the solvent is within large channels it crystallizes at 155 K, as identified by a sharp change in the increase of unit-cell volume upon temperature increase. In contrast, if a similar amount of solvent is confined to narrow channels and/or individual cavities it does not crystallize in the studied temperature range. It is concluded that the solvent in large channels behaves similarly to bulk water, whereas when confined to narrow channels it is mainly protein-associated. The analogy with the behaviour of pure bulk water provides circumstantial evidence that only solvent in large channels undergoes a glass transition in the 100--180 K temperature range. These studies reveal that flash-cooled protein crystals are arrested in a metastable state up to at least 155 K, thus providing an upper temperature limit for their storage and handling. The results are pertinent to the development of rational crystal annealing procedures and to the study of temperature-dependent radiation damage to proteins. Furthermore, they suggest an experimental paradigm for studying the correlation between solvent behaviour, protein dynamics and protein function.

Acetylcholinesterase↗
PubMed2001

Specific protein dynamics near the solvent glass transition assayed by radiation-induced structural changes.

The nature of the dynamical coupling between a protein and its surrounding solvent is an important, yet open issue. Here we used temperature-dependent protein crystallography to study structural alterations that arise in the enzyme acetylcholinesterase upon X-ray irradiation at two temperatures: below and above the glass transition of the crystal solvent. A buried disulfide bond, a buried cysteine, and solvent exposed methionine residues show drastically increased radiation damage at 155 K, in comparison to 100 K. Additionally, the irradiation-induced unit cell volume increase is linear at 100 K, but not at 155 K, which is attributed to the increased solvent mobility at 155 K. Most importantly, we observed conformational changes in the catalytic triad at the active site at 155 K but not at 100 K. These changes lead to an inactive catalytic triad conformation and represent, therefore, the observation of radiation-inactivation of an enzyme at the atomic level. Our results show that at 155 K, the protein has acquired--at least locally--sufficient conformational flexibility to adapt to irradiation-induced alterations in the conformational energy landscape. The increased protein flexibility may be a direct consequence of the solvent glass transition, which expresses as dynamical changes in the enzyme's environment. Our results reveal the importance of protein and solvent dynamics in specific radiation damage to biological macromolecules, which in turn can serve as a tool to study protein flexibility and its relation to changes in a protein's environment.

Acetylcholinesterase↗
PubMed2001

Fluoride concentration of bottled drinking waters.

The use of bottled water and beverages may be a significant source of systemic fluoride and can therefore be considered as a risk factor for dental fluorosis in young children. The aim of this study was to determine the fluoride content of commercially available bottled drinking waters and to report on the accuracy of the labelling of fluoride concentration. Thirty brands of bottled water, classified as either spring (N = 19) or mineral (N = 11) water were evaluated. A fluoride ions-elective and a fluoride reference electrode were used to measure the fluoride concentrations. The average reading for each brand was compared with the fluoride content printed on the label. Only 56.7% (N = 17) of brands tested mention the fluoride concentration on the label, but 73.3% (N = 22) had a tested fluoride concentration of less than 0.3 ppm. Of the 8 brands testing higher than 0.3 ppm fluoride, 1 did not have the fluoride concentration labelled, while for another the tested fluoride concentration was much higher than the concentration printed on the label. When prescribing fluoride supplements, dentists should be aware of the fluoride content of bottled waters used by child patients, especially brands with a concentration higher than 0.3 ppm.

Child↗
PubMed2001

Evaluation of Atraumatic Restorative Treatment restorations and sealants under field conditions.

Conventional restorative procedures require the use of dental materials, expensive equipment, instruments and electricity. This makes dental restorations costly and limits the availability and accessibility of the service to areas where these facilities are available. The new philosophy of restorative care requires minimal intervention and advocates early interception of the caries process through the use of glass-ionomers as sealants and as restorations. This new approach makes dental care accessible to all members of society in developing countries. The aim of this study was to evaluate the use of Atraumatic Restorative Treatment (ART) restorations and sealants under field conditions. Both were assessed for their retentive properties and the development of secondary caries adjacent to the procedures. A longitudinal study design was followed. ART restorations and sealants were assessed at intervals of 6 and 12 months. After 12 months 56.5% of ART restorations were still present in the primary dentition and 84% in the permanent dentition. Of these 85.1% had no defects in the primary and 78.6% no defects in the permanent teeth. 72.3% of primary and 92% of permanent teeth on which ART restorations were placed had no caries after 1 year. Even though most of the sealants were absent due to poor retention after 12 months (89.6%), 98.9% of the teeth where the sealant was initially placed had no caries. Only 6.3% of sealants present had marginal defects. The ART technique is strongly recommended for the management of small, occlusal carious lesions in primary and permanent teeth.

Child↗
PubMed2001

Caries prediction factors in children with primary dentition.

Previous studies have indicated an association between the dmft and the lactobacilli counts in small children. This study evaluated and compared a number of salivary factors that could have an effect on caries progression in two groups of children with primary dentition (group 1 = 3-6 years; group II = 9 years). The average dmft score was higher for group II. The dmft score of group I consisted mainly of a large dt component, while in group II a large ft component was found. Lactobacilli were present in 44.83% of group I and in 77.27% of group II. Significant positive correlations were found for group I between the dt component of the dmft and lactobacilli count (P < 0.05, r = 0.48) as well as the total dmft and lactobacilli count (P < 0.05, r = 0.45). Significant positive correlations were found for group II between the dmft and lactobacilli count (P < 0.05, r = 0.39) and the plaque index and lactobacilli count (P < 0.05, r = 0.31). Significant correlations between the dmft and the prevalence of lactobacilli in the oral cavity were also indicated (group I: P < 0.05, r = 0.45; group II: P < 0.05, r = 0.36). Significant correlations confirmed the association of lactobacilli with the caries process and indicated the reliability of lactobacilli counts to determine caries activity. Correlations between the dmft and the prevalence of lactobacilli in the oral cavity indicated the possibility of an excellent but simple test for the prediction of caries susceptibility in children.

Buffers↗
PubMed2001

Trends in treatment performed in the Phelophepa Dental Clinic: 1995-2000.

Mobile clinics are a cost-effective method of meeting the dental needs of rural communities in South Africa. Phelophepa, the first primary health care train of its kind world-wide, provides eye care, education, medicine, basic health care and since June 1995 dental treatment to rural communities. All services are rendered by students under supervision of qualified staff. The aim of this study was to analyse and report the data for treatment performed in the dental clinic from June 1995 to May 2000. During its first five years of operation, dental services were provided at 183 towns in all provinces except Gauteng. Of the 42,073 patients treated during this time (an average of 229.9 per town), 67.4% were adults. 71.3% of the 103,283 procedures performed were extractions, 15.7% could be classified as preventive with the remaining 13% as restorative procedures. The average value of the service provided to each patient was R218.53. The exposure of dental, dental therapy and oral hygiene students to rural areas of South Africa serves the important purpose of sensitising students to the realities of oral diseases in these communities.

Adult↗
PubMed2000

Identification of the collagen-binding site of the von Willebrand factor A3-domain.

Von Willebrand factor (vWF) is a multimeric glycoprotein that mediates platelet adhesion and thrombus formation at sites of vascular injury. vWF functions as a molecular bridge between collagen and platelet receptor glycoprotein Ib. The major collagen-binding site of vWF is contained within the A3 domain, but its precise location is unknown. To localize the collagen-binding site, we determined the crystal structure of A3 in complex with an Fab fragment of antibody RU5 that inhibits collagen binding. The structure shows that RU5 recognizes a nonlinear epitope consisting of residues 962-966, 981-997, and 1022-1026. Alanine mutants were constructed of residues Arg(963), Glu(987), His(990), Arg(1016), and His(1023), located in or close to the epitope. Mutants were expressed as fully processed multimeric vWF. Mutation of His(1023) abolished collagen binding, whereas mutation of Arg(963) and Arg(1016) reduced collagen binding by 25-35%. These residues are part of loops alpha3beta4 and alpha1beta2 and alpha-helix 3, respectively, and lie near the bottom face of the domain. His(1023) and flanking residues display multiple conformations in available A3-crystal structures, suggesting that binding of A3 to collagen involves an induced-fit mechanism. The collagen-binding site of A3 is located distant from the top face of the domain where collagen-binding sites are found in homologous integrin I domains.

Alanine↗
PubMed2000

Specific chemical and structural damage to proteins produced by synchrotron radiation.

Radiation damage is an inherent problem in x-ray crystallography. It usually is presumed to be nonspecific and manifested as a gradual decay in the overall quality of data obtained for a given crystal as data collection proceeds. Based on third-generation synchrotron x-ray data, collected at cryogenic temperatures, we show for the enzymes Torpedo californica acetylcholinesterase and hen egg white lysozyme that synchrotron radiation also can cause highly specific damage. Disulfide bridges break, and carboxyl groups of acidic residues lose their definition. Highly exposed carboxyls, and those in the active site of both enzymes, appear particularly susceptible. The catalytic triad residue, His-440, in acetylcholinesterase, also appears to be much more sensitive to radiation damage than other histidine residues. Our findings have direct practical implications for routine x-ray data collection at high-energy synchrotron sources. Furthermore, they provide a direct approach for studying the radiation chemistry of proteins and nucleic acids at a detailed, structural level and also may yield information concerning putative "weak links" in a given biological macromolecule, which may be of structural and functional significance.

Acetylcholinesterase↗