Shielding of the A1 domain by the D'D3 domains of von Willebrand factor modulates its interaction with platelet glycoprotein Ib-IX-V.

Soluble von Willebrand factor (VWF) has a low affinity for platelet glycoprotein (GP) Ibalpha and needs immobilization and/or high shear stress to enable binding of its A1 domain to the receptor. The previously described anti-VWF monoclonal antibody 1C1E7 enhances VWF/GPIbalpha binding and recognizes an epitope in the amino acids 764-1035 region in the N-terminal D'D3 domains. In this study we demonstrated that the D'D3 region negatively modulates the VWF/GPIb-IX-V interaction; (i) deletion of the D'D3 region in VWF augmented binding to GPIbalpha, suggesting an inhibitory role for this region, (ii) the isolated D'D3 region inhibited the GPIbalpha interaction of a VWF deletion mutant lacking this region, indicating that intramolecular interactions limit the accessibility of the A1 domain, (iii) using a panel of anti-VWF monoclonal antibodies, we next showed that the D'D3 region is in close proximity with the A1 domain in soluble VWF but not when VWF was immobilized; (iv) destroying the epitope of 1C1E7 resulted in a mutant VWF with an increased affinity for GPIbalpha.

Attachment of water-soluble proteins to the surface of (magnetizable) phospholipid colloids via NeutrAvidin-derivatized phospholipids.

The present work describes the incorporation of a functionalized phospholipid derivative into the phospholipid bilayer of both classical small unilamellar vesicles and recently developed magnetoliposomes, resulting in unique biocolloid structures onto which peripheral water-soluble enzymes can be immobilized on the surfaces. In the first part of this work, a synthesis protocol is outlined for a universal membrane anchor for water-soluble proteins. Dioleoylphosphatidylethanolamine-N-dodecanyl was used as the starting lipid molecule.

Immobilised peptide displaying phages as affinity ligands. Purification of lactoferrin from defatted milk.

An affinity purification procedure for the direct purification of lactoferrin from defatted (skimmed) milk has been developed. The procedure is based on using selected phage clones expressing a peptide with high binding affinity for lactoferrin which were covalently coupled to macroporous poly(dimethylacrylamide) monolithic column. Large pore size (10-100 microm) of macroporous poly(dimethylacrylamide) makes it possible to couple long (1 microm) phage particles as ligands without any risk of blocking the monolithic column.

Tryptophan to phenylalanine substitutions allow differentiation of short- and long-range conformational changes during denaturation of goat alpha-lactalbumin.

To test the occurrence of local particularities during the unfolding of Ca2+-loaded goat alpha-lactalbumin (GLA) we replaced Trp60 and -118, either one or both, by Phe. In contrast with alternative studies, our recombinant alpha-lactalbumins are expressed in Pichia pastoris and do not contain the extra N-terminal methionine. The substitution of Trp60 leads to a reduction of the global stability.

The interaction of LEDGF/p75 with integrase is lentivirus-specific and promotes DNA binding.

We have previously shown that the p75 isoform of the transcriptional co-activator lens epithelium-derived growth factor (LEDGF) interacts tightly with human immunodeficiency virus (HIV)-1 integrase (IN) and is essential for nuclear targeting of this protein in human cells (Cherepanov, P., Maertens, G., Proost, P.

Receptor-mediated biological responses are prolonged using hydrophobized ligands.

Hormone-receptor interactions occur following three-dimensional diffusion of the ligand to the membrane-embedded receptor. However, prior hydrophobization of the ligand might restrict its movement to two dimensions along the membrane surface, and the biological response might therefore be modulated. This idea was tested using the C-terminal nonapeptide, CCK9, of the satiating hormone, cholecystokinin (CCK).

A probe for capture and Fe3+-induced conformational change of lactoferrin selected from phage displayed peptide libraries.

Linear pentadecamer and cyclic hexamer peptide phage libraries were used to isolate phage clones with binding affinity toward lactoferrins purified from human and bovine milk. Phage clones with high specificity toward lactoferrin were selected with different binding strengths depending on the sequence of the peptide displayed by the phage. Phages coated to a microtiterplate were able to capture lactoferrin from crude milk samples without prior treatment.

Simmondsin: effects on meal patterns and choice behavior in rats.

Simmondsin, a glycoside from jojoba meal, decreases food intake after oral administration. The present experiments are designed to clarify the mechanism of simmondsin's anorectic activity. The meal pattern analysis shows that simmondsin supplementation at different doses results in a dose-dependent food intake reduction, which is more pronounced after prior simmondsin experience.

Antibacterial and antifungal properties of alpha-helical, cationic peptides in the venom of scorpions from southern Africa.

Two novel pore-forming peptides have been isolated from the venom of the South-African scorpion Opistophtalmus carinatus. These peptides, designated opistoporin 1 and 2, differ by only one amino acid and belong to a group of alpha-helical, cationic peptides. For the first time, a comparison of the primary structures of alpha-helical pore-forming peptides from scorpion venom was undertaken.

Photoexcitation of tryptophan groups induces reduction of two disulfide bonds in goat alpha-lactalbumin.

Illumination of goat alpha-lactalbumin (GLA) with 280 or 295 nm light results in tryptophan-mediated photolysis of disulfide bonds within the protein. The photolysis is not dependent on the absence or presence of Ca(2+) and is observed as well on illumination of native and of partially unfolded GLA. However, photolysis of native GLA results in a partial unfolding of the protein.

The frequency width of symmetric modes of an aluminum plate: manifestation of a complex slowness on a frequency spectrum.

The evolution of the experimental frequency width of symmetric modes of an aluminum plate is studied as a function of the angle of incidence below the first critical angle. It is found that the frequency width predicted by resonant scattering theory, corrected for the directivity of emitter and receiver, generally explains the experimental frequency width well. However, large discrepancies remain for the frequency width of the S1 mode at angles of incidence larger than 9 degrees.

Structural basis for difference in heat capacity increments for Ca(2+) binding to two alpha-lactalbumins.

Thermodynamic parameters for the unfolding of as well as for the binding of Ca(2+) to goat alpha-lactalbumin (GLA) and bovine alpha-lactalbumin (BLA) are deduced from isothermal titration calorimetry in a buffer containing 10 mM Tris-HCl, pH 7.5 near 25 degrees C. Among the different parameters available, the heat capacity increments (Delta C(p)) offer the most direct information for the associated conformational changes of the protein variants.

Teratological studies in defatted jojoba meal-supplemented rats.

To look for possible developmental effects in the offspring of jojoba meal-treated Wistar rats, and to distinguish between the effects of reduced food intake and the specific developmental effects of jojoba meal itself, mated female rats were divided into three groups of 20 rats. They received during gestation: (a) normal rodent food (control group); (b) normal rodent food supplemented with 3% defatted jojoba meal (jojoba group); or (c) normal rodent food pair-fed with the jojoba group (pair-fed group). The jojoba meal group showed approximately 30% inhibition of food intake.

Preparation and Characterization of a Phospholipid Membrane-Bound Tetrapeptide That Corresponds to the C-Terminus of the Gastrin/Cholecystokinin Hormone Family.

The present work deals with the synthesis of a hydrophobized peptide and its localization at the membrane surface, after its incorporation into phospholipid vesicles. The tetrapeptide, Trp-Met-Asp-Phe-NH(2), which corresponds to the C-terminus of the cholecystokinin/gastrin hormone family, is conjugated to N-glutaryldioleoylphosphatidylethanolamine using a carbodiimide-catalyzed reaction method. Sonication of the lipophilized hormone in the presence of dimyristoylphosphatidylcholine results in a strong sequestration of the conjugate in the artificial membrane structures that are formed.

The perturbations of the native state of goat alpha-lactalbumin induced by 1,1'-bis(4-anilino-5-naphthalenesulfonate) are Ca2+-dependent.

In this work we have studied the interaction of the hydrophobic fluorescent probe 1,1'-bis(4-anilino-5-naphthalenesulfonate) (bis-ANS), with the native state of apo- and Ca2+-bound goat alpha-lactalbumin (GLA). In 10 mM Tris-HCl, pH 7.5, at 4 degrees C in 2 mM EGTA as well as at 37 degrees C in 2 mM Ca2+, the native protein is close to its thermal transition.

Comparison of the effects of simmondsin and cholecystokinin on metabolism, brown adipose tissue and the pancreas in food-restricted rats.

In this study, we investigated the analogies between the physiological effects of simmondsin, a satiety-inducing glycoside extracted from jojoba seeds, and the gastro-intestinal satiation peptide, cholecystokinin. The effects of intraperitoneal injection of the biological active CCK-octapeptide on the pancreas, interscapular brown adipose tissue, growth performance and energy metabolism in normal-fed, severely food intake-restricted (50 % of normal food intake) or moderately food intake-restricted (65 % of normal food intake) growing rats were compared to the effects of 0.25 % simmondsin mixed in the food, inducing moderate food intake reduction (65 % of normal) in rats.

Reproductive performance of rats treated with defatted jojoba meal or simmondsin before or during gestation.

The effects on food intake, growth and reproductive performance parameters of defatted jojoba meal and pure simmondsin, an extract from jojoba meal, were compared in female Wistar rats. Rats fed 0.15% simmondsin or 3% defatted jojoba meal (equivalent to 0.

Simple two-step procedure for the preparation of highly active pure equine milk lysozyme.

A fast, simple two-step purification scheme is presented for the isolation of lysozyme at a high yield from equine milk. In the first step, fluidized bed technology, using the Streamline system, was exploited. In the following step, advantage was taken of Ca(2+)-induced conformational changes to obtain a pure, high specific activity, enzyme fraction by hydrophobic interaction chromatography.

Devazepide reverses the anorexic effect of simmondsin in the rat.

Simmondsin, a glycoside extracted from jojoba meal (Simmondsia chinensis), causes a reduction in food intake after oral administration. To investigate the mechanism by which simmondsin reduces food intake, fasted and free-feeding rats were given simmondsin-supplemented food and simultaneously injected with devazepide, a specific antagonist of peripheral-type cholecystokinin receptors (CCKA receptors). In free-feeding rats, supplementation of food with 0.

Complexes of the polyamines spermine, spermidine and putrescine with alpha-lactalbumins.

The effects of polyamines on the spectral properties and thermal stability of different alpha-lactalbumins were measured. Addition of millimolar concentrations of spermine to the Ca(2+)-free (apo) form of bovine or goat alpha-lactalbumin resulted in spectral shifts, in both the far- and near-ultraviolet ranges, similar to those induced by Ca2+ binding. Fluorescence emission spectra of tryptophan residues underwent a pronounced blue shift, concomitant with a decrease in quantum yield.