Effects of soybean saponin on protease hydrolyses of beta-lactoglobulin and alpha-lactalbumin.
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Effects of Soybean Saponin on Protease Hydrolyses of beta-Lactoglobulin and alpha-Lactalbumin The effects of soybean saponin on tryptic and chymotryptic hydrolyses of whey proteins were evaluated. beta-lactoglobulin and alpha-lactalbumin became more sensitive to both trypsin and chymotrypsin by interacting with saponin in contrast to serum albumin. Soybean saponin was shown to have different effects on various proteins according to their nature. (+info)
Thermal unfolding and refolding of beta-lactoglobulin. An intrinsic andextrinsic fluorescence study.
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The conformational features of beta-lactoglobulin, refolded by cooling from a thermally perturbed state, has been characterized by intrinsic and extrinsic fluorescence measurements on the protein. It is found that even at 85-90 degrees C, beta-lactoglobulin does not completely lose its folded structure. The unfolding and refolding of beta-lactoglobulin as observed through intrinsic tryptophan fluorescence is nearly reversible because the native beta-lactoglobulin and its refolded form, following heating and cooling, show nearly identical tryptophan fluorescence properties. However, the fluorescence properties of an extrinsic probe 1-anilino 8-naphthalene sulfonic acid (ANS) for the native and refolded forms are quite different from each other. Significant increase in fluorescence intensity and blue shifts in emission maxima of ANS bound to refolded beta-lactoglobulin is observed compared to that of the native form. Our results indicate that beta-lactoglobulin, refolded after heating to above 70 degrees C, has deep hydrophobic pockets which can be accessed by ANS. These pockets are either nonexistent or inaccessible to ANS in native beta-lactoglobulin. The opening of the central cavity collapses at pH close to the isoelectric pH of the protein. This indicates that electrostatic repulsion is necessary to keep this access open. (+info)
Breast cancer-specific expression of the Candida albicans cytosine deaminase gene using a transcriptional targeting approach.
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We constructed a series of adenoviral (Ad) vectors that express the Candida albicans cytosine deaminase (CD) suicide gene under the transcriptional control of either the human alpha-lactalbumin (ALA) or ovine beta-lactoglobulin (BLG) promoter (Ad.ALA.CD and Ad.BLG.CD, respectively). The Ad.ALA.CD and the Ad.BLG.CD vectors converted the prodrug 5-fluorocytosine (5-FC) to the toxic nucleotide analog 5-fluorouracil in a breast cancer cell-specific manner, with a conversion rate of 40% and 52% in T47D cells and 50% and 41% in MCF7 cells, respectively. No significant conversion (< or =3%) was observed in an immortalized nontumorigenic breast epithelial cell line (MCF10A) and a human osteosarcoma cell line (U2OS). Adenovirus vector-based prodrug conversion of the 5-FC in T47D and MCF7 in the presence of 1 mg/mL of 5-FC led to cytotoxicity that resulted in a nearly complete cell death (> or =90%) after 5 days, whereas MCF10A and U2OS cells remained resistant (< or =10%). Nude mice harboring T47D-derived breast tumors that were injected intratumorally (i.t.) with therapeutic adenovirus vectors at a dose of 2 x 10(8) plaque-forming units and treated systemically with 5-FC at a concentration of 500 mg/kg/day showed a marked reduction in tumor mass within 30 days when compared with animals that received vector alone. Animal survival was significantly prolonged after 72 days in mice treated with therapeutic vectors in conjunction with prodrug when compared with control animals. These preclinical data are sufficiently promising to warrant further studies of this transcriptional targeting approach to breast cancer treatment. (+info)
IgE antibodies to Dermatophagoides pteronyssinus (Housedust mite), Aspergillus fumigatus, and beta-lactoglobulin in sudden infant death syndrome.
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The prevalence of serum IgE antibodies to Dermatophagoides pteronyssinus (house-dust mite), Aspergillus fumigatus, and bovine beta-lactoglobulin was significantly greater in cases of sudden infancy death (S.I.D.) than in a control group of infants of the same age range. This difference was most pronounced with D. pteronyssinus antibodies, which suggests that hypersensitivity to house-dust mite may be a factor in the aetiology of S.I.D. in Western Australia. Both the S.I.D. and control infants had similar serum concentrations of immunoglobulins G, M, and E but IgA levels were significantly higher in the control group. (+info)
Inhibition of Escherichia coli by bovine colostrum and post-colostral milk. II. The bacteriostatic effect of lactoferrin on a serum susceptible and serum resistant strain of E. coli.
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Two strains of Escherichia coli were inhibited by complement-inactivated cow serum and to a lesser extent by precolostral calf serum devoid of specific antibodies. They were not inhibited by undiluted colostral whey or milk but colostral whey became bacteriostatic after dialysis or dilution in Kolmer saline and addition of precolostral calf serum or lactoferrin. The inhibition in all these fluids was due to iron-binding proteins (transferrin or lactoferrin). Undiluted dialysed milk was not inhibitory because of its low content of lactoferrin but became inhibitory after addition of 1 mg/ml of lactoferrin. The lack of inhibition in undiluted whey is due to the high concentration of citrate in colostral whey (and milk) and it is suggested that citrate competes with the iron-binding proteins for iron and makes it availabe to the bacteria. Addition of bicarbonate, which is required for the binding of iron by transferrin and lactoferrin, can overcome the effect of citrate; hence, the bacteriostatic effect of cow serum and precolostral calf serum is due to the presence of both transferrin and bicarbonate as well as the low lefel of citrate. (+info)
Characterization and isolation of intermediates in beta-lactoglobulin heat aggregation at high pH.
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The early stages of heat induced aggregation at 67.5 degrees C of beta-lactoglobulin were studied by combined static light scattering and size exclusion chromatography. At all conditions studied (pH 8.7 without salt and pH 6.7 with or without 60 mM NaCl) we observe metastable heat-modified dimers, trimers, and tetramers. These oligomers reach a maximum in concentration at about the time when large aggregates (1000-4000 kg/mol) appear, after which they decline in concentration. By isolating the oligomers it was demonstrated that they rapidly form aggregates upon heating in the absence of monomeric protein, showing that these species are central to the aggregation process. To our knowledge this is the first time that intermediates in protein aggregation have been isolated. At all stages of aggregation the dominant oligomer was the heat-modified dimer. Whereas the heat-modified oligomers are formed at a higher rate at pH 8.7 than at pH 6.7, the opposite is the case for the formation of aggregates from the metastable oligomers indicating cross-linking via disulfide bridges for the oligomers and noncovalent interaction in the formation of the aggregates. The data suggest that an aggregate nucleus is formed from four oligomers. For protein concentrations of 10 or 20 g/l a heat-modified monomer can be observed until about the time when the maximum in concentration appears of the heat-modified dimer. The disappearance of this heat-modified monomer correlates to the formation of dimers (trimers and tetramers). (+info)
Bovine beta-lactoglobulin: interaction studies with palmitic acid.
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Bovine beta-lactoglobulin (BLG) in vivo has been found complexed with fatty acids, especially palmitic and oleic acid. To elucidate the still unknown structure-function relationship in this protein, the interactions between 13C enriched palmitic acid (PA) and BLG were investigated by means of one-, two-, and three-dimensional NMR spectroscopy in the pH range 8.4-2.1. The NMR spectra revealed that at neutral pH the ligand is bound within the central cavity of BLG, with the methyl end deeply buried within the protein. The analysis of 13C spectra of the holo protein revealed the presence of conformational variability of bound PA carboxyl end in the pH range 8.4-5.9, related to the Tanford transition. The release of PA starts at pH lower than 6.0, and it is nearly complete at acidic pH. This finding is relevant in relation to the widely reported hypothesis that this protein can act as a transporter through the acidic gastric tract. Ligand binding and release is shown to be completely reversible over the entire pH range examined, differently from other fatty acid binding proteins whose behavior is analyzed throughout the paper. The mode of interaction of BLG is compatible with the proposed function of facilitating the digestion of milk fat during the neonatal period of calves. (+info)
Effect of pH on the structure and aggregation of human glycodelin A. A comparison with beta-lactoglobulin A.
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The effect of pH on the structure of glycodelin A (GdA) and of beta-lactoglobulin A (beta-LgA) has been investigated by means of circular dichroism, steady state fluorescence, synchrotron radiation small angle X-ray scattering (SR-SAXS) and gel permeation chromatography. The comparison between GdA and beta-LgA shows that, at pH 7.0, both proteins are dimers with an extended content of beta-sheet conformation, but pH 2.0 and 9.0 yield a different secondary, tertiary and quaternary structural organisation. Whilst beta-LgA is a monomer, that conserves beta-sheet conformation at pH 2.0 and 9.0, GdA has a stable dimeric structure at alkaline pH, but at pH 2.0 increases its alpha-helix content and it aggregates soon. SR beam has been used to perform SAXS comparative measurements of the two proteins. SR-SAXS data provide the radius of gyration and the radii of the cross-section and of the thickness. GdA aggregation at acid pH has been characterised by calculating the distance distribution function (P(r)). Isoelectric focusing and chromatofocusing data show a different charge distribution on the surfaces of the two proteins, supporting the hypothesis that the presence of oligosaccharides deeply influences the conformational state and the aggregation process of GdA at different pH values. In particular, the presence of sialic acid residues, within the oligosaccharide moiety of the GdA, might be responsible for the differences observed between the two proteins. (+info)