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Biomedical subjects

M De Maeyer

Publications and source records attributed to M De Maeyer.

At least 19 recordsLinked to original sources
PubMed2006

Sources of saturated fatty acids in Belgian adolescents' diet: implications for the development of food-based dietary guidelines.

The objectives of the present study are to describe the dietary sources of total fat and of saturated fatty acids (SFA) and to formulate food-based dietary guidelines for SFA in Belgian adolescents. A random sample of 13-18-year-old adolescents was drawn from secondary schools in the region of Ghent. A 7 d estimated food record method was used to quantify nutrient and food intake. The average daily SFA intake is 4 % above the recommended 10 % of the total energy contribution. The most important contributors of SFA on food group level were 'fats, oils and savoury sauces', 'meat and meat products', 'sugar, confectionery, sweet fillings and sauces', 'cheese', 'milk and milk products' and 'bread, rusk and breakfast rolls'. On food subgroup level 'fresh meat', 'high-fat margarine' and 'high-fat cheese' had the highest contribution to SFA intake in all adolescents. Adolescents with a low SFA intake (lowest tertile) were compared with adolescents with a high intake (highest tertile). In the lowest tertile the intake of total fat and MUFA was significantly lower than in the highest tertile, while the intake of total carbohydrates, mono- and disaccharides and complex carbohydrates was significantly higher. Overall, the high-fat cheese intake is significantly lower in the lowest tertile, while the fruit intake is higher. The present analysis shows that the nutritional profile of Belgian adolescents could be potentially improved by decreasing the portion sizes of fresh meat (in boys), high-fat margarine, high-fat cheese and reducing intake of commercially prepared baked goods and processed foods, including fast foods.

Adolescent↗
PubMed2005

Iron intake and dietary sources of iron in Flemish adolescents.

OBJECTIVE: To investigate the dietary iron intake and food sources of iron in Flemish adolescents. DESIGN: Cross-sectional survey; dietary assessment method: a 7-day estimated food record. SETTING: Private and public secondary schools in Ghent, a city in the Dutch-speaking part of Belgium. SUBJECTS: A total of 341 adolescents (129 boys and 212 girls), 13-18 y, randomly selected by a multistage clustered sampling technique. RESULTS: The mean total iron intake (s.d.) for boys was 13.4 (+/- 2.91) mg/day and for girls 10.1 (+/- 2.79) mg/day. A proportion of 38.8% of the boys and 99.5% of the girls had a mean total iron intake below the Belgian Recommended Dietary Allowance and 3.1% of the boys and 71.2% of the girls below the British Estimated Average Requirement. When bioavailable iron intake is considered, 84.5% of the boys and only 16.5% of the girls met the age-specific requirement. The food groups with the highest mean proportional contribution to total iron intake in both males and females were bread, meat and meat products, cereals and potatoes. A comparison of adolescents from the highest tertile of iron intake (mg/day) with adolescents from the lowest tertile showed a significantly higher energy-adjusted intake of brown bread and a significantly lower intake of soft drinks in the former group in both boys and girls. A significantly higher energy-adjusted intake of breakfast cereals in adolescents of the highest tertile than those of the lowest tertile was seen in girls only. Analyses in consumers only did not change this overall picture. CONCLUSIONS: One can conclude that the mean iron intake of Flemish girls is considerably lower than the current recommendations. An increased iron intake in this subgroup of the population is therefore advisable.

Adolescent↗
PubMed2003

Molecular mechanisms of mild and moderate hemophilia A.

Mutations responsible for mild/moderate hemophilia A were extensively characterized over the last 15 years and more than 200 mutations have been identified. However, most of the molecular mechanisms responsible for the reduced factor (F)VIII levels in patients' plasma were determined only recently. Recent progresses in the study of the FVIII molecule three-dimensional structure provided a major insight for understanding molecular events leading to mild/moderate hemophilia A. This allowed prediction of mutations impairing FVIII folding and intracellular processing, which result in reduced FVIII secretion. Mutations potentially slowing down FVIII activation by thrombin were also identified. A number of mutations were also predicted to result in altered stability of activated FVIII. Biochemical analyses allowed identification of mutations reducing FVIII production. Mutations impairing FVIII stability in plasma, by reducing FVIII binding to von Willebrand factor (VWF) were also characterized. Defects in FVIII activity, notably slow activation by thrombin, or abnormal interaction with FIXa, were also recently demonstrated. Biochemical analysis of FVIII variants provided information regarding the structure/function relationship of the FVIII molecule and validated predictions of the three-dimensional structure of the molecule. These observations also contributed to explain the discrepant activities recorded for some FVIII variants using different types of FVIII assays. Altogether, the study of the biochemical properties of FVIII variants and the evaluation of the effects of mutations in three-dimensional models of FVIII identified molecular mechanisms potentially explaining reduced FVIII levels for a majority of patients with mild/moderate hemophilia A. It is expected that these studies will improve diagnosis and treatment of this disease.

Factor IXa↗
PubMed2001

Critical role of glutamic acid 202 in the enzymatic activity of stromelysin-1 (MMP-3).

To test the hypothesis that Glu202, adjacent to the His201 residue that participates in the coordination of Zn(2+) in matrix metalloproteinase-3 (MMP-3 or stromelysin-1), plays a role in its enzymatic activity it was substituted with Ala, Lys or Asp by site-specific mutagenesis. Wild-type proMMP-3, proMMP-3(E202A), proMMP-3(E202K) and proMMP-3(E202D) were expressed in Escherichia coli and purified to apparent homogeneity. Whereas 33-kDa wild-type proMMP-3 (consisting of the propeptide and catalytic domains) was quantitatively converted to 24-kDa active MMP-3 by treatment with p-aminophenyl-mercuric acetate (APMA), proMMP-3(E202A) and proMMP-3 (E202K) were fully resistant to APMA and proMMP-3 (E202D) was quantitatively converted into a 14-kDa species. In contrast, treatment with plasmin quantitatively converted the wild-type and the three mutant proMMP-3 moieties into the corresponding 24-kDa MMP-3 moieties. Biospecific interaction analysis revealed comparable affinity for binding to plasminogen of wild-type and mutant proMMP-3 (K(a) of 2.6-6.3 x 10(6) M(-1)) or MMP-3 (K(a) of 33-58 x 10(6) M(-1)) moieties. The affinity for binding to single-chain urokinase-type plasminogen activator (scu-PA) was also similar for wild-type and mutant proMMP-3 (K(a) of 5.0-6.9 x 10(6) M(-1)) or MMP-3 (K(a) of 37-72 x 10(6) M(-1)) moieties. However, MMP-3(E202A) and MMP-3(E202K) did not hydrolyze plasminogen whereas MMP-3(E202D) showed an activity of 20--30% of wild-type MMP-3. All three mutants were inactive towards scu-PA under conditions where this was quantitatively cleaved by wild-type MMP-3. Furthermore, MMP-3(E202A) and MMP-3(E202K) were inactive toward a fluorogenic substrate and MMP-3 (E202D) displayed about 15% of the activity of wild-type MMP-3. Taken together, these data suggest that Glu202 plays a crucial role in the enzymatic activity of MMP-3.

Alanine↗
PubMed2000

Polyethylene glycol-derivatized cysteine-substitution variants of recombinant staphylokinase for single-bolus treatment of acute myocardial infarction.

BACKGROUND: Thrombolytic therapy of acute myocardial infarction (AMI) is evolving toward bolus administration. Derivatization of proteins with polyethylene glycol (PEG) may reduce their clearance. METHODS AND RESULTS: A staphylokinase (SakSTAR) variant with 12 amino acid substitutions to reduce its antigenicity, SakSTAR (K35A, E65Q, K74R, E80A, D82A, T90A, E99D, T101S, E108A, K109A, K130T, K135R), and with Ser in position 3 mutated into Cys (code SY161), was derivatized with maleimide-PEG with M:(r) of 5,000 (P5), 10,000 (P10), or 20,000 (P20). The PEGylated variants recognized only one third of the antibodies elicited with wild-type SakSTAR in AMI patients. In experimental animals, plasma clearances were reduced 2. 5- to 5-fold with P5, 5- to 20-fold with P10, and 20-fold with P20, and bolus injection induced pulmonary plasma clot lysis at doses inversely related to their clearance. Intravenous bolus injection of 5 mg of the P5, P10, or P20 variants in AMI patients was associated with plasma half-lives (t(1/2alpha)) of 13, 30, and 120 minutes and clearances of 75, 43, and 8 mL/min, respectively, compared with 3 minutes and 360 mL/min for SakSTAR. Injection of 5 mg P5 variant restored TIMI-3 flow within 60 minutes in 14 of 18 AMI patients (78%, 95% CI 55% to 91%) and of 2.5 mg in 7 of 11 patients (63%, 95% CI 35% to 85%), both in the absence of fibrinogen degradation. The immunogenicity of the variants was significantly (P:<0.002) reduced. CONCLUSIONS: The staphylokinase variant SY161-P5, derivatized with one linear polyethylene glycol molecule of M:(r) 5000, is a promising fibrin-selective agent for single-bolus coronary thrombolysis.

Acute Disease↗
PubMed1999

A capping domain for LRR protein interaction modules.

Leucine-rich repeats (LRR) are protein interaction modules which are present in a large number of proteins with diverse functions. We describe here a novel motif (16-19 residues) downstream of the last, incomplete, LRR in a subfamily of LRR proteins. In the U2A' spliceosomal protein, this motif is folded into a cap that shields the hydrophobic core of the LRRs from the solvent. Modelling of the LRR-cap in the imidazoline-1 candidate receptor, using the known structure of U2A' as template, showed a conservation of the basic structural features.

Amino Acid Sequence↗
PubMed1999

Guiding a docking mode by phage display: selection of correlated mutations at the staphylokinase-plasmin interface.

During co-evolution of interacting proteins, functionally disruptive mutations on one side of the interface may be compensated by local amino acid changes on the other to restore binding affinity. This information can be useful for geometry-based docking approaches by reducing the translational and rotational space available to the proteins. Here, we demonstrate that correlated mutations at a protein-protein interface can be rapidly identified by selecting a phage-displayed library of a randomly mutated component of the complex for complementation of mutations that decreased binding in the interacting partner. This approach was used to deduce the binding mode of staphylokinase (Sak), a 15.5 kDa "indirect" plasminogen activator on microplasmin (microPli), the 28 kDa serine protease domain of plasmin. Biopanning indicated that residues Arg94 and Gly174 in microPli are located in close proximity to Glu75 and the Glu88:Ile128 pair in Sak, respectively. The coupled mutations Glu94<-->Lys75 reversed and Gly174<-->Lys88:Val128 introduced a salt bridge, whereby the binding affinities (with coupling energies of 1.8 to 2.3 kcal mol-1, respectively) and the plasminogen activation ability of the mutated complexes were partially restored. These findings suggested a unique docking mode of Sak at the western rim of the active-site cleft of microPli, that is in agreement with the structure of the Sak-microPli complex as recently derived by other methods.

Amino Acid Substitution↗
PubMed1999

Detection of SV40 like viral DNA and viral antigens in malignant pleural mesothelioma.

This study investigated the presence of simian vacuolating virus 40 (SV40) deoxyribonucleic acid (DNA) in malignant pleural mesothelioma, non-neoplastic mesothelium and pleural carcinoma metastasis and correlated these data with immunohistochemistry for SV40 viral antigens. The novel Primed In Situ (PRINS) method was applied to detect the presence of SV40 DNA in situ in tissue sections of malignant mesothelioma (n = 25), non-neoplastic mesothelium (n = 30) and pleural carcinoma metastasis (n = 30). Immunohistochemistry with an SV40-specific antibody was applied for detection of the SV40 viral antigen in the same material. SV40 DNA and expression of one of the viral proteins (small t-antigen) was found in approximately 60% of the investigated mesothelioma cases in contrast to non-neoplastic mesothelium and carcinoma metastasis that were negative for both SV40 DNA and SV40 viral antigens. These results suggest that simian vacuolating virus 40 deoxyribonucleic acid may be biologically active as there was also immunoreactivity for simian vacuolating virus 40 viral antigen in those cases positive for simian vacuolating virus 40 deoxyribonucleic acid with the primed in situ reaction. Simian vacuolating virus 40 viral deoxyribonucleic acid and antigens may be potential markers for neoplastic mesothelium that may prove useful in the rather difficult histopathological differential diagnosis between malignant mesothelioma and reactive mesothelium or pleural carcinoma.

Antigens, Viral↗
PubMed1999

Structural and functional basis of plasminogen activation by staphylokinase.

Staphylokinase (Sak), a 15.5-kDa bacterial protein, forms a complex with human plasmin, which in turn activates other plasminogen molecules to plasmin. Three recombinant DNA-based approaches, (i) site directed substitution with alanine, (ii) search for proximity relationships at the complex interface, and (iii) active-site accessibility to protease inhibitors have been used to deduce a coherent docking model of the crystal structure of Sak on the homology-based model of microplasmin (microPli), the serine protease domain of plasmin. Sak binding on microPli is primarily mediated by two surface-exposed loops, loops 174 and 215, at the rim of the active-site cleft, while the binding epitope of Sak on microPli involves several residues located in the flexible NH2-terminal arm and in the five-stranded mixed beta-sheet. Several Sak residues located within the unique alpha-helix and the beta2 strand do not contribute to the binding epitope but are essential to induce plasminogen activating potential in the Sak:microPli complex. These residues form a topologically distinct activation epitope, which, upon binding of Sak to the catalytic domain of microPli, protrudes into a broad groove near the catalytic triad of microPli, thereby generating a competent binding pocket for micro-plasminogen (microPlg), which buries approximately 2500 A of the Sak:microPli complex upon binding. This structural and functional model may serve as a template for the design of improved Sak-derived thrombolytic agents. Following the completion and presentation of the present study, the deduced Sak:microPli:microPlg complex was fully confirmed by X-ray crystallography, which further illustrates the power and potential of the present approach.

Bacteriophages↗
PubMed1998

Arginine 719 in human plasminogen mediates formation of the staphylokinase:plasmin activator complex.

Staphylokinase (Sak), a 16-kDa bacterial protein, forms a 1:1 stoichiometric complex with the serine proteinase domain of human plasmin, which in turn converts other plasminogen molecules into plasmin. To identify amino acid residues critical for generating the Sak:plasmin activator complex, alanine-scanning mutagenesis was performed on phage-displayed micro-plasminogen (microPlg). Substitution of Arg719 with Ala [microPlg(R719A)] disrupted complex formation, although the sensitivity of phage-displayed microPlg(R719A) to activation by urokinase and the amidolytic activity of the micro-plasmin derivative [microPli(R719A)] remained unaffected. Likewise, the soluble microPlg(R719A) molecule did not generate a functional activator complex with Sak, whereas quantitative activation into plasmin was obtained upon incubation with either urokinase or the Sak:plasmin complex. Real-time biospecific affinity measurements revealed that the Arg --> Ala substitution at position 719 increased the equilibrium dissociation constant between microPlg(R719A) and Sak from 46 nM to 1 microM, primarily by reducing the association rate constant. Arg719 has recently also been implied in the functional complex formation between human plasmin and streptokinase [Dawson, K. M., Marshall, J. M., Raper, R. H., Gilbert, R. J., and Ponting, C. P. (1994) Biochemistry 33, 12042-12047.], suggesting that both bacterial cofactors may share common structural and/or mechanistic aspects for plasminogen activation.

Amino Acid Substitution↗
PubMed1997

Trematode myoglobins, functional molecules with a distal tyrosine.

The myoglobins of two trematodes, Paramphistomum epiclitum and Isoparorchis hypselobagri, were isolated to homogeneity. The native molecules are monomeric with Mr 16,000-17,000 and pI 6.5-7.5. In each species, at least four different globin isoforms occur. Primary structure was determined at the protein level. The globin chains contain 147 amino acid residues. Although major determinants of the globin fold are conserved, characteristic substitutions are present. A Tyr residue occurs at the helical positions B10 and E7 (distal position). This is confirmed by NMR measurements (Zhang, W., Rashid, K. A., Haque, M., Siddiqi, A. H., Vinogradov, S. N., Moens, L. & La Mar, G. N. (1997) J. Biol. Chem. 272, 3000-3006). A distal Tyr normally provokes oxidation of the iron atom and the inability to bind oxygen, whereas a Tyr-B10 is indicative for a high oxygen affinity. In contrast, trematode myoglobins are functional molecules with a high oxygen affinity. Molecular modeling predicts two possible positions for the aromatic ring of Tyr-E7: one being outside the heme pocket making it freely accessible to the ligand and one within the heme pocket potentially able to form a second hydrogen bond with the iron-bound oxygen. A hydrogen bond between Tyr-B10 and the bound oxygen as in the Ascaris hemoglobin is predicted as well. The predicted structure may explain the high oxygen affinity of the trematode myoglobins.

Amino Acid Sequence↗
PubMed1997

All in one: a highly detailed rotamer library improves both accuracy and speed in the modelling of sidechains by dead-end elimination.

BACKGROUND: About a decade ago, the concept of rotamer libraries was introduced to model sidechains given known mainchain coordinates. Since then, several groups have developed methods to handle the challenging combinatorial problem that is faced when searching rotamer libraries. To avoid a combinatorial explosion, the dead-end elimination method detects and eliminates rotamers that cannot be members of the global minimum energy conformation (GMEC). Several groups have applied and further developed this method in the fields of homology modelling and protein design. RESULTS: This work addresses at the same time increased prediction accuracy and calculation speed improvements. The proposed enhancements allow the elimination of more than one-third of the possible rotameric states before applying the dead-end elimination method. This is achieved by using a highly detailed rotamer library allowing the safe application of an energy-based rejection criterion without risking the elimination of a GMEC rotamer. As a result, we gain both in modelling accuracy and in computational speed. Being completely automated, the current implementation of the dead-end elimination prediction of protein sidechains can be applied to the modelling of sidechains of proteins of any size on the high-end computer systems currently used in molecular modelling. The improved accuracy is highlighted in a comparative study on a collection of proteins of varying size for which score results have previously been published by multiple groups. Furthermore, we propose a new validation method for the scoring of the modelled structure versus the experimental data based upon the volume overlap of the predicted and observed sidechains. This overlap criterion is discussed in relation to the classic RMSD and the frequently used +/- 40 degrees window in comparing chi 1 and chi 2 angles. CONCLUSIONS: We have shown that a very detailed library allows the introduction of a safe energy threshold rejection criterion, thereby increasing both the execution speed and the accuracy of the modelling program. We speculate that the current method will allow the sidechain prediction of medium-sized proteins and complex protein interfaces involving up to 150 residues on low-end desktop computers.

Algorithms↗
PubMed1997

Dead-end based modeling tools to explore the sequence space that is compatible with a given scaffold.

The dead-end elimination algorithm has proven to be a powerful tool in protein homology modeling since it allows one to determine rapidly the global minimum-energy conformation (GMEC) of an arbitrarily large collection of side chains, given fixed backbone coordinates. After introducing briefly the necessary background, we focus on logic arguments that increase the efficacy of the dead-end elimination process. Second, we present new theoretical considerations on the use of the dead-end elimination method as a tool to identify sequences that are compatible with a given scaffold structure. Third, we initiate a search for properties derived from the computed GMEC structure to predict whether a given sequence can be well packed in the core of a protein. Three properties will be considered: the nonbonded energy, the accessible surface area, and the extent by which the GMEC side-chain conformations deviate from a locally optimal conformation.

Amino Acid Sequence↗
PubMed1997

Computation of the binding of fully flexible peptides to proteins with flexible side chains.

Docking algorithms play an important role in the process of rational drug design and in understanding the mechanism of molecular recognition. An important determinant for successful docking is the extent to which the configurational space (including conformational changes) of the ligand/receptor system is searched. Here we describe a new, combinatorial method for flexible docking of peptides to proteins that allows full rotation around all single bonds of the peptide ligand and around those of a large set of receptor side chains. We have simulated the binding of several viral peptides to murine major histocompatibility complex class I H-2Kb. In addition, we have explored the limits of our method by simulating a complex between calmodulin and an 18-residue long helical peptide from calmodulin-dependent protein kinase IIalpha. The calculated peptide conformations generally matched well with the X-ray structures. Essential information about local flexibility and about residues that are responsible for strong binding was obtained. We have frequently observed considerable side-chain flexibility during the simulations, showing the need for a flexible treatment of the receptor. Our method may also be useful whenever the receptor side-chain conformation is not available or uncertain, as illustrated by the docking of an H-2Kb binding nonapeptide to the receptor structure taken from an octapeptide/H-2Kb complex.

Alanine↗
PubMed1997

Theoretical and algorithmical optimization of the dead-end elimination theorem.

The dead-end elimination theorem has proved to be a powerful method to reduce the theoretically accessible conformational space when modeling protein side chains by using a rotameric representation of possible conformations. In this work, theoretical details about variants to the original criterion are discussed. We also provide information on how the equations can be algorithmically implemented in such a way that both computational performance and structural accuracy are optimized. In addition, we discuss the theoretical and practical aspects of three new methods called the "bottom line theorem", dead-end elimination assisted by local modeling and a combinatorial search combined with conventional dead-end elimination. It is shown that the algorithm in its current from enables the determination of the global minimum energy side chain conformation of large proteins on a time scale of hours while for small proteins of up to 30 residues the calculations are done on a time scale of seconds. The latter opens a way to combine a main chain sampling algorithm with the dead-end elimination method to locally model entire fragments of a protein chain.

Algorithms↗
PubMed1995

Enhanced dead-end elimination in the search for the global minimum energy conformation of a collection of protein side chains.

Although the conformational states of protein side chains can be described using a library of rotamers, the determination of the global minimum energy conformation (GMEC) of a large collection of side chains, given fixed backbone coordinates, represents a challenging combinatorial problem with important applications in the field of homology modelling. Recently, we have developed a theoretical framework, called the dead-end elimination method, which allows us to identify efficiently rotamers that cannot be members of the GMEC. Such dead-ending rotamers can be iteratively removed from the system under study thereby tracking down the size of the combinatorial problem. Here we present new developments to the dead-end elimination method that allow us to handle larger proteins and more extensive rotamer libraries. These developments encompass (i) a procedure to determine weight factors in the generalized dead-end elimination theorem thereby enhancing the elimination of dead-ending rotamers and (ii) a novel strategy, mainly based on logical arguments derived from the logic pairs theorem, to use dead-ending rotamer pairs in the efficient elimination of single rotamers. These developments are illustrated for proteins of various sizes and the flow of the current method is discussed in detail. The effectiveness of dead-end elimination is increased by two orders of magnitude as compared with previous work. In addition, it now becomes feasible to use extremely detailed libraries. We also provide an appendix in which the validity of the generalized dead-end criterion is shown. Finally, perspectives for further applications which may now become within reach are discussed.

Aprotinin↗