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PubMed2005

Strain hardening and fracture of heat-set fractal globular protein gels.

Non-linear mechanical behavior at large shear deformation was been investigated for heat-set beta-lactoglobulin gels at pH 7 and 0.1 M NaCl using both oscillatory shear and shear flow. These gels have a self-similar structure at length scales smaller than the correlation length of the gel with fractal dimension d(f)=2. Strain hardening is observed that can be well described using the model proposed by Gisler et al. [T.C. Gisler, R.C. Ball, D.A. Weitz, Phys. Rev. Let. 82 (1999) 1064] for fractal colloidal gels. The increase of the shear modulus normalized by the low strain value (G(0)) is independent of G(0). For weak gels the elasticity increases up to a factor of ten, while for strong gels the increase is very small. At higher deformation irreversible fracture occurs, which leads eventually to macroscopic failure of the gel. For weak gels formed at low concentrations the deformation at failure is about 2, independent of the shear modulus. For strong gels fracture occurs at approximately constant stress (2 x 10(3) Pa).

Fractals↗
PubMed2006

The adsorption and unfolding kinetics determines the folding state of proteins at the air-water interface and thereby the equation of state.

Unfolding of proteins has often been mentioned as an important factor during the adsorption process at air-water interfaces and in the increase of surface pressure at later stages of the adsorption process. This work focuses on the question whether the folding state of the adsorbed protein depends on the rate of adsorption to the interface, which can be controlled by bulk concentration. Therefore, the adsorption of proteins with varying structural stabilities at several protein concentrations was studied using ellipsometry and surface tensiometry. For beta-lactoglobulin the adsorbed amount (Gamma) needed to reach a certain surface pressure (Pi) decreased with decreasing bulk concentration. Ovalbumin showed no such dependence. To verify whether this difference in behavior is caused by the difference in structural stability, similar experiments were performed with cytochrome c and a destabilized variant of this protein. Both proteins showed identical Pi-Gamma, and no dependence on bulk concentration. From this work it was concluded that unfolding will only take place if the kinetics of adsorption is similar or slower than the kinetics of unfolding. The latter depends on the activation energy of unfolding (which is in the order of 100-300 kJ/mol), rather than the free energy of unfolding (typically 10-50 kJ/mol).

Adsorption↗
PubMed2006

Coupling between polysaccharide gelation and micro-phase separation of globular protein clusters.

The effect of gelation of the polysaccharide phase on the phase separation was investigated for mixtures of anionic polysaccharide (kappa-carrageenan) and globular protein (beta-lactoglobulin) clusters at pH 7 well above the iso-electric point. Gelation of kappa-carrageenan was induced by cooling in the presence of KCl. In the liquid state the protein clusters phase-separate into relatively dense micro-domains. When the polysaccharide phase gelled during cooling, the turbidity of the systems decreased dramatically. Light scattering experiments showed that the density of the micro-domains decreased, while microscopy showed that the number and size was not strongly modified. It is concluded that smaller protein clusters leave the micro-domains when kappa-carrageenan gels. The effect could be reversed by reheating the samples and thus melting the gel and was observed for repeated heating and cooling cycles. The effect of gelation on phase separation decreases with increasing polysaccharide concentration and with ageing of the liquid mixture. The latter is caused by the formation of bonds between the protein clusters in the micro-domains that slowly reinforce with time.

Carrageenan↗
PubMed2004

Use of immunodotting to select the desorption agent for immunochromatography.

In immunochromatography, a technique of increasing use, the sample containing the antigen (Ag) to be purified or determined is introduced into a chromatographic column containing an antibody (Ab) bound to the packing material. The antigen is retained based on antigen-antibody recognition. To reuse the immunocolumn for subsequent assays, the antigen has to be eluted without causing irreversible damage of antibodies. Selection of conditions for performing immunochromatography is usually made by trial and error. This way of working is time consuming and it may ruin the column. In this article, the feasibility of using immunodotting to select the conditions to be employed in one immunochromatographic assay is shown. An immunodotting method is developed to select the best desorption agent for an enzyme-linked immunoaffinity chromatography (ELIAC) assay to determine beta-lactoglobulin (beta-LG). The effect of several factors on the immunodotting performance is studied. The way of performing solvent exchange to treat the antibody with different solutions considered as potential desorption agents to check their effect is shown. Effectiveness of the solution chosen by immunodotting (4 M MgCl(2) in 20 mM Tris, pH 5.9) as desorption agent is demonstrated by immunochromatographic assays. The immunodotting and solvent exchange methods developed should be useful to choose solvents and conditions for any other kind of assay.

Antibodies↗
PubMed2005

The binding of thioflavin-T to amyloid fibrils: localisation and implications.

Amyloid fibrils are a polymeric form of protein, involving a continuous beta-sheet with the strands perpendicular to the long axis of the fibril. Although typically implicated in diseases such as Alzheimer's disease and the transmissible spongiform encephalopathies, non disease-associated protein can also be converted into amyloid fibrils. Traditionally, amyloid fibrils are identified via the use of specific dyes such as Congo red and thioflavin-T, although their specificity is ill understood. Recently, solutions of bovine insulin and bovine beta-lactoglobulin have been found to form spherulites, micron-sized spherical structures containing radially arranged amyloid fibrils. When studied by confocal microscopy using polarised laser light and thioflavin-T, a consistent pattern of emission, rather than a uniform disc, was observed. This suggests the dye binds in a specific, regular fashion to amyloid fibrils. Confocal microscopy studies of thioflavin-T aligned in stretched poly-vinyl alcohol films showed that the dye dipole excitation axis lies parallel to the long molecular axis. Therefore, thioflavin-T binds to amyloid fibrils such that their long axes are parallel. We propose binding occurs in 'channels' that run along the length of the beta-sheet. Steric interactions between dye molecules and side chains indicate why thioflavin-T fluoresces more intensely when bound to amyloid fibrils and can explain why this interaction with amyloid fibrils is specific, but with varying efficiency.

Amyloid↗
PubMed2006

Species-specific cell-matrix interactions are essential for differentiation of alveoli like structures and milk gene expression in primary mammary cells of the Cape fur seal (Arctocephalus pusillus pusillus).

Few models are in place for analysis of extreme lactation patterns such as that of the fur seals which are capable of extended down regulation of milk production in the absence of involution. During a 10-12 month lactation period, female fur seals suckle pups on shore for 2-3 days, and then undertake long foraging trips at sea for up to 28 days, resulting in the longest intersuckling bouts recorded. During this time the mammary gland down regulates milk production. We have induced Cape fur seal (Arctocephalus pusillus pusillus) mammary cells in vitro to form mammospheres up to 900 microm in diameter, larger than any of their mammalian counterparts. Mammosphere lumens were shown to form via apoptosis and cells comprising the cellular boundary stained vimentin positive. The Cape fur seal GAPDH gene was cloned and used in RT-PCR as a normalization tool to examine comparative expression of milk protein genes (alphaS2-casein, beta-lactoglobulin and lysozyme C) which were prolactin responsive. Cape fur seal mammary cells were found to be unique; they did not require Matrigel for rapid mammosphere formation and instead deposited their own matrix within 2 days of culture. When grown on Matrigel, cells exhibited branching/stellate morphogenesis highlighting the species-specific nature of cell-matrix interactions during morphological differentiation. Matrix produced in vitro by cells did not support formation of human breast cancer cell line, PMC42 mammospheres. This novel model system will help define the molecular pathways controlling the regulation of milk protein expression and species specific requirements of the extracellular matrix in the cape fur seal.

Animals↗
PubMed2006

Modulation of the infant immune responses by the first pertussis vaccine administrations.

Many efforts are currently made to prepare combined vaccines against most infectious pathogens, that may be administered early in life to protect infants against infectious diseases as early as possible. However, little is known about the general immune modulation induced by early vaccination. Here, we have analyzed the cytokine secretion profiles of two groups of 6-month-old infants having received as primary immunization either a whole-cell (Pw) or an acellular (Pa) pertussis vaccine in a tetravalent formulation of pertussis-tetanus-diphtheria-poliomyelitis vaccines. Both groups of infants secreted IFN-gamma in response to the Bordetella pertussis antigens filamentous haemagglutinin and pertussis toxin, and this response was correlated with antigen-specific IL-12p70 secretion, indicating that both pertussis vaccines induced Th1 cytokines. However, Pa recipients also developed a strong Th2-type cytokine response to the B. pertussis antigens, as noted previously. In addition, they induced Th2-type cytokines to the co-administrated antigen tetanus toxoïd, as well as to the food antigen beta-lactoglobulin. Furthermore, the general cytokine profile of the Pa recipients was strongly Th2-skewed at 6 months, as indicated by the cytokines induced by the mitogen phytohaemagglutinin. These data demonstrate that the cytokine profile of 6-month-old infants is influenced by the type of formulation of the pertussis vaccine they received at 2, 3 and 4 months of life. Large prospective studies would be warranted to evaluate the possible long-term consequences of this early modulation of the cytokine responses in infants.

Adhesins, Bacterial↗
PubMed2001

The specificity of monoglyceride-protein interactions and mechanism of the protein induced L(beta) to coagel phase transition.

This study aims at gaining insight into the specificity and molecular mechanism of monoglyceride-protein interactions. We used beta-lactoglobulin (beta-LG) and lysozyme as model proteins and both monostearoylglycerol and monopalmitoylglycerol as defined gel phase monoglycerides. The monoglycerides were used in different combinations with the two negatively charged amphiphiles dicetylphosphate and distearylphosphate. The interactions were characterized using the monolayer technique, isothermal titration calorimetry, (2)H-nuclear magnetic resonance (NMR) using deuterium labelled monoglycerides and freeze fracture electron microscopy (EM). Our results show that lysozyme inserts efficiently into all monolayers tested, including pure monoglyceride layers. The insertion of beta-LG depends on the lipid composition of the monolayer and is promoted when the acylchains of the negatively charged amphiphile are shorter than that of the monoglyceride. The binding parameters found for the interaction of beta-LG and lysozyme with monoglyceride bilayers were generally similar. Moreover, in all cases a large exothermic binding enthalpy was observed which was found to depend on the nature of the monoglycerides but not of the proteins. (2)H-NMR and freeze fracture EM showed that this large enthalpy results from a protein mediated catalysis of the monoglyceride L(beta) to coagel phase transition. The mechanism of this phase transition consists of two steps, an initial protein mediated vesicle aggregation step which is followed by stacking and probably fusion of the bilayers.

Freeze Fracturing↗
PubMed1986

Differential cytolysis of murine spleen, bone-marrow and leukemia cells by melittin reveals differences in membrane topography.

L1210 leukemia cells are 2-4 fold more sensitive to the cytolytic effects of melittin, the membrane-active toxin of bee venom, than normal DBA/2 mouse spleen and bone-marrow cells. Lysis of the normal cells was abolished when either 75 mM galactosamine, glucosamine or 100 microM beta-lactoglobulin was added to the melittin-cell reaction, but lysis of the leukemia cells was unaffected. The amino-groups appeared necessary for blocking melittin-mediated lysis since glucose, galactose and the N-acetyl derivatives were not inhibitory. Bone-marrow cells were more readily protected from lysis than spleen cells. Since melittin-inhibitor complexes were not detected by gel chromatography and the inhibitor could be added to the cell suspension after melittin, the evidence suggests that bone-marrow cells are rich in membrane binding sites for carbohydrates that decrease in mature spleen cells and are virtually absent after neoplastic transformation.

Animals↗
PubMed2001

Hydration and protein folding in water and in reverse micelles: compressibility and volume changes.

The partial specific volume and adiabatic compressibility of proteins reflect the hydration properties of the solvent-exposed protein surface, as well as changes in conformational states. Reverse micelles, or water-in-oil microemulsions, are protein-sized, optically-clear microassemblies in which hydration can be experimentally controlled. We explore, by densimetry and ultrasound velocimetry, three basic proteins: cytochrome c, lysozyme, and myelin basic protein in reverse micelles made of sodium bis (2-ethylhexyl) sulfosuccinate, water, and isooctane and in aqueous solvents. For comparison, we use beta-lactoglobulin (pI = 5.1) as a reference protein. We examine the partial specific volume and adiabatic compressibility of the proteins at increasing levels of micellar hydration. For the lowest water content compatible with complete solubilization, all proteins display their highest compressibility values, independent of their amino acid sequence and charge. These values lie within the range of empirical intrinsic protein compressibility estimates. In addition, we obtain volumetric data for the transition of myelin basic protein from its initially unfolded state in water free of denaturants, to a folded, compact conformation within the water-controlled microenvironment of reverse micelles. These results disclose yet another aspect of the protein structural properties observed in membrane-mimetic molecular assemblies.

Animals↗
PubMed2001

Detergents as probes of hydrophobic binding cavities in serum albumin and other water-soluble proteins.

As an extension of our studies on the interaction of detergents with membranes and membrane proteins, we have investigated their binding to water-soluble proteins. Anionic aliphatic compounds (dodecanoate and dodecylsulfate) were bound to serum albumin with high affinity at nine sites; related nonionic detergents (C12E8 and dodecylmaltoside) were bound at seven to eight sites, many in common with those of dodecanoate. The compounds were also bound in the hydrophobic cavity of beta-lactoglobulin, but not to ovalbumin. In addition to the generally recognized role of the Sudlow binding region II of serum albumin (localized at the IIIA subdomain) in fatty acid binding, quenching of the fluorescence intensity of tryptophan-214 by 7,8-dibromododecylmaltoside and 12-bromododecanoate also implicate the Sudlow binding region I (subdomain IIA) as a locus for binding of aliphatic compounds. Our data document the usefulness of dodecyl amphipathic compounds as probes of hydrophobic cavities in water-soluble proteins. In conjunction with recent x-ray diffraction analyses of fatty acid binding as the starting point we propose a new symmetrical binding model for the location of nine high-affinity sites on serum albumin for aliphatic compounds.

Allosteric Regulation↗
PubMed2002

Spectroscopic studies on the interaction of a water soluble porphyrin and two drug carrier proteins.

The interaction of meso-tetrakis(p-sulfonatophenyl)porphyrin (TSPP) sodium salt to human serum albumin and beta-lactoglobulin was studied by steady-state and dynamic fluorescence at different pH of aqueous solutions. The formation of TSPP J-aggregates and a noncovalent TSPP-protein complex was monitored by fluorescence titrations, which depend on pH and on the protein nature and concentration. The complex between TSPP and protein displays a heterogeneous equilibrium with large changes in the binding strength versus pH. The large reduction of the effective binding constant from pH 2 to 7 suggests that electrostatic interactions are a major contribution to the binding of TSPP to the aforementioned proteins. TSPP aggregates and TSPP-protein complex exhibit circular dichroism induced by the presence of the protein. Circular dichroism spectra in the ultraviolet region show that the secondary structure of both proteins is not extensively affected by the TSPP presence. Protein-TSPP interaction was also examined by following the intrinsic fluorescence of the tryptophan residues of the proteins. Fluorescence quenching by acrylamide and TSPP itself also point to small changes on the protein tertiary structure and a critical distance R(0) approximately 56 A, between tryptophan and bound porphyrin, was estimated using the long distance Förster-type energy transfer formalism.

Acrylamides↗
PubMed2002

Interaction of proteins in solution from small-angle scattering: a perturbative approach.

In this work an improved methodology for studying interactions of proteins in solution by small-angle scattering is presented. Unlike the most common approach, where the protein-protein correlation functions g(ij)(r) are approximated by their zero-density limit (i.e., the Boltzmann factor), we propose a more accurate representation of g(ij)(r) that takes into account terms up to the first order in the density expansion of the mean-force potential. This improvement is expected to be particularly effective in the case of strong protein-protein interactions at intermediate concentrations. The method is applied to analyze small-angle x-ray scattering data obtained as a function of the ionic strength (from 7 to 507 mM) from acidic solutions of beta-lactoglobulin at the fixed concentration of 10 gl(-1). The results are compared with those obtained using the zero-density approximation and show significant improvement, particularly in the more demanding case of low ionic strength.

Animals↗
PubMed2003

Low-frequency modes of peptides and globular proteins in solution observed by ultrafast OHD-RIKES spectroscopy.

The low-frequency (1-200 cm(-1)) vibrational spectra of peptides and proteins in solution have been investigated with ultrafast optical heterodyne-detected Raman-induced Kerr-effect spectroscopy (OHD-RIKES). Spectra have been obtained for di-L-alanine (ALA(2)) and the alpha-helical peptide poly-L-alanine (PLA) in dichloroacetic acid solution. The poly-L-alanine spectrum shows extra amplitude compared to the di-L-alanine spectrum, which can be explained by the secondary structure of the former. The globular proteins lysozyme, alpha-lactalbumin, pepsin, and beta-lactoglobulin in aqueous solution have been studied to determine the possible influence of secondary or tertiary structure on the low-frequency spectra. The spectra of the globular proteins have been analyzed in terms of three nondiffusive Brownian oscillators. The lowest frequency oscillator corresponds to the so-called Boson peak observed in inelastic neutron scattering (INS). The remaining two oscillators are not observed in inelastic neutron scattering, do therefore not involve significant motion of hydrogen atoms, and may be associated with delocalized backbone torsions.

Alanine↗
PubMed1972

Photolysis accompanying peptide absorption in proteins. Electrophoresis and optical rotary dispersion studies.

Exposure of proteins and polypeptides to ultraviolet radiation below 240 nm produces peptide cleavage which may or may not be accompanied by observable changes in conformation and optical rotary dispersion (ORD) properties, depending on the stability of the secondary and tertiary structure of the macromolecule under the experimental conditions. Helical and coiled forms of poly-L-glutamic acid undergo degradation at similar rates but only the helical form shows a significant change in rotatory properties. The helical form of poly-L-lysine, but neither the coiled nor beta forms, shows a change in [alpha](233) on irradiation at 233 nm. beta-Lactoglobulin shows essentially no change in [alpha](233) on irradiation in either dilute salt solution or 4 M urea at room temperature; however, in 4 M urea at 56 degrees C a large change occurs. A model is developed which shows that studies of the effect of radiation on ORD properties may be useful in providing information on possible intermediate steps in protein denaturation. The method is illustrated with results on bovine plasma albumin. A quantum yield, 4.3 x 10(-3) moles/einstein, was obtained for peptide cleavage in this protein at 225 nm. These studies, based on gel electrophoresis, also showed that the fragments produced are essentially random, suggesting that transfer of energy from aromatic residues is not an important contributor to the peptide photolysis. Possible errors which could arise in ORD and other studies involving intense ultraviolet radiation are considered.

Animals↗
PubMed2002

Transfer of orlistat through oil-water interfaces.

The transfer of radiolabelled orlistat ([14C]orlistat), a potent gastrointestinal lipase inhibitor, through an oil-water interface from a single oil droplet to an aqueous phase was investigated, using an oil drop tensiometer. The absolute transfer fluxes were found to be very low, even in the presence of micellar concentrations of bile salts, which increased their values from 0.2 to 2.5 and 6.5 pmol cm(-2) min(-1) in the presence of 0, 4 and 15 mM NaTDC, respectively. Adding either a lipid emulsion or pure human pancreatic lipase (HPL) or human serum albumin or beta-lactoglobulin had no effect on the flux of transfer of orlistat. The presence of colipase or a mixture of colipase and HPL was found, however, to reduce the flux of orlistat transfer, probably because it partly covered the single oil drop surface, even in the presence of bile salts. Using a finely emulsified system, we investigated the partitioning of orlistat between the aqueous and oil phases, in the absence or presence of bile salts above their CMC (4 mM NaTDC, final concentration). Under these emulsified conditions, orlistat was found to be mostly associated with the oil phase, since more than 98.8% of the total radioactivity was recovered after decantation with the oil phase. The low transfer rates of orlistat, as well as its partitioning coefficient between the oil and the aqueous phases, should help us to better understand the inhibitory effects of orlistat on lipid digestion in humans.

Animals↗
PubMed1997

Tests of vaginal microbicides in the mouse genital herpes model.

Microbicide candidates were selected that have demonstrated activity against sperm or sexually transmitted disease pathogens in vitro, and the efficacy of these agents for preventing vaginal transmission of genital herpes infection was evaluated in the progestin-treated mouse. Each agent was delivered to the vaginas of mice approximately 20 sec prior to delivering a highly infectious herpes simplex virus-2 inoculum. The following agents provided significant protection: anti-HSV monoclonal antibodies III-174 and HSV8, modified bovine beta-lactoglobulin (beta-69), carrageenan, concanavalin A, chlorhexidine, dextran sulfate (average molecular weight 8,000 and 500,000), fucoidan, neem, nonoxynol-9, polystyrene sulfonate, and povidone-iodine. Two agents, gramicidin and heparan sulfate, though highly effective in vitro, were not protective in vivo at the doses tested.

Animals↗
PubMed1999

Potentiation of anaphylaxis in guinea pig ileal mucosa by a selective delta-opioid receptor agonist.

Immediate hypersensitivity reactions in the intestinal mucosa evoke active chloride secretion which enhances the elimination of luminal antigens. The prosecretory actions of histamine and other soluble mediators of anaphylaxis are mediated by submucosal neurons, as are the antisecretory actions of opioid antidiarrheal medications. We tested the hypothesis that the selective delta-opioid receptor agonist [D-Pen2, D-Pen5]enkephalin (DPDPE) alters anaphylaxis-associated ileal anion secretion in vitro. Sheets of ileal mucosa with attached submucosa from guinea pigs sensitized to cow's milk were mounted in Ussing chambers under short-circuit conditions. Mucosal sheets responded to the serosal application of the milk protein, beta-lactoglobulin, with a rapid rise in transepithelial short-circuit current (Isc); in contrast, the egg protein, ovalbumin, was without effect. Pretreatment of tissues with the neuronal conduction blocker, saxitoxin, or the H1 histamine receptor antagonist, diphenhydramine, but not the opioid receptor antagonist, naloxone, significantly reduced mucosal responses to antigen. [D-Pen-2, D-Pen5]enkephalin (0.1 microM, serosal addition) decreased baseline Isc, but potentiated mucosal responses to antigen; its effects were abolished in tissues pretreated with naloxone. These results suggest that immediate hypersensitivity reactions in the guinea pig ileal mucosa are mediated by submucosal neural circuits that are phasically modulated by both mast cell products and opioids.

Analgesics↗