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PubMed2006

The adsorbed conformation of globular proteins at the air/water interface.

External reflection FTIR spectroscopy and surface pressure measurements were used to compare conformational changes in the adsorbed structures of three globular proteins at the air/water interface. Of the three proteins studied, lysozyme, bovine serum albumin and beta-lactoglobulin, lysozyme was unique in its behaviour. Lysozyme adsorption was slow, taking approximately 2.5 h to reach a surface pressure plateau (from a 0.07 mM solution), and led to significant structural change. The FTIR spectra revealed that lysozyme formed a highly networked adsorbed layer of unfolded protein with high antiparallel beta-sheet content and that these changes occurred rapidly (within 10 min). This non-native secondary structure is analogous to that of a 3D heat-set protein gel, suggesting that the adsorbed protein formed a highly networked interfacial layer. Albumin and beta-lactoglobulin adsorbed rapidly (reaching a plateau within 10 min) and with little change to their native secondary structure.

Adsorption↗
PubMed2000

Cord blood levels of immunoglobulin G subclass antibodies to food and inhalant allergens in relation to maternal atopy and the development of atopic disease during the first 8 years of life.

BACKGROUND: Factors that either protect from or enhance the development of atopic disease appear to be acting early in life. The gestational environment, including maternal immune responses, such as transplacentally transferred immunoglobulin (Ig) G antibodies to allergens, may be of importance in this respect, since allergen-specific immunity has been demonstrated to develop in utero. OBJECTIVE: To evaluate the relation between cord blood IgG subclass antibodies to allergens, maternal atopy and development of atopic disease in the children. MATERIAL AND METHODS: The study group comprised a cohort of 96 children participating in a prospective study up to 8 years of age. Cord blood IgG subclass antibodies to ovalbumin, beta-lactoglobulin, Bet v 1 and cat dander were analysed by ELISA. RESULTS: The levels of all IgG subclass antibodies to ovalbumin and rBet v 1 were higher in newborn infants with an atopic mother, as compared with babies with nonatopic mothers. IgG1 antibody levels to cat and IgG4 antibody levels to beta-lactoglobulin and cat were also higher in atopic than in nonatopic mothers, whereas the other subclass antibody levels to those allergens were similar. High levels of cord blood IgG antibodies to cat and birch, but not to the food allergens, were associated with less atopic symptoms in the children during the first 8 years of life. Moreover, children who developed IgE antibodies to cat had lower levels of IgG antibodies to that allergen at birth. CONCLUSIONS: High levels of cord blood IgG subclass, especially IgG4, antibodies to food and inhalant allergens are associated with maternal atopy. High levels of IgG antibodies to inhalant, but not food, allergens are associated with less development of atopy in the children.

Administration, Inhalation↗
PubMed2006

Evaluation of intrinsic compressibility of proteins by molecular dynamics simulation.

Molecular dynamics simulation has been performed on five native proteins in water to evaluate their intrinsic isothermal compressibilities beta(T,int). To identify physical factors contributing to protein compressibility, a general method is presented for analyzing the compressibility of mechanically inhomogeneous systems. The value of beta(T,int) varies with protein species considerably: beta-lactoglobulin (14.15 x 10(-2) GPa(-1)) is more than twice as compressible as ribonuclease A (6.77 x 10(-2) GPa(-1)). Beta-lactoglobulin and myoglobin (13.95 x 10(-2) GPa(-1)) have similar values of beta(T,int), but the mechanisms responsible for them are significantly different. The volume fluctuations of internal cavities and the magnitudes of the crosscorrelation between them are the key factors determining beta(T,int) of proteins. Though the volume fractions of internal cavity for the five studied proteins are nearly equal to one another, the mean cavity compressibilities beta(T,cav) vary considerably with protein species and range from 0.35 to 0.69 GPa(-1), which are much smaller than those of normal organic liquids such as methanol, ethanol, and benzene and close to that of glycerol (0.55 GPa(-1)), a strongly associated liquid.

Animals↗
PubMed2002

B-cell epitopes as a screening instrument for persistent cow's milk allergy.

BACKGROUND: Cow's milk is one of the most common causes of food allergy in the first years of life. We recently defined IgE-binding epitopes of all 6 major cow's milk proteins (alpha(s1)-, alpha(s2)-, beta-, and kappa-casein; alpha-lactalbumin; and beta-lactoglobulin) and had some evidence suggesting that IgE antibodies from patients with persistent cow's milk allergy (CMA) recognize different epitopes on cow's milk proteins than do those from patients who were likely to outgrow their allergy. OBJECTIVE: In this study we sought to assess whether recognition of IgE antibodies of certain epitopes of cow's milk proteins would clearly separate the patients with life-long CMA from those who will become clinically tolerant to cow's milk. METHODS: According to the known IgE-binding regions of cow's milk proteins, 25 decapeptides of alpha(s1)-casein, alpha(s2)-casein, kappa-casein, alpha-lactalbumin, and beta-lactoglobulin, comprising the core epitopes, were synthesized on a cellulose-derivatized membrane. Sera from 10 patients with persistent CMA and 10 patients who subsequently outgrew their milk allergy were used to investigate the differences in epitope recognition. RESULTS: Five IgE-binding epitopes (2 on alpha(s1)-casein, 1 on alpha(s2)-casein, and 2 on kappa-casein) were not recognized by any of the patients with transient CMA but showed binding by the majority of the patients with persistent allergy. The presence of IgE antibodies against at least 1 of 3 epitopes (amino acid [AA] 123-132 on alpha(s1)-casein, AA 171-180 on alpha(s2)-casein, and AA 155-164 on kappa-casein) identified all patients with persistent CMA. CONCLUSIONS: The presence of IgE antibodies to distinct allergenic epitopes of cow's milk proteins can be used as a marker of persistent CMA. Prospective studies are needed to investigate the usefulness of these informative epitopes in predicting life-long CMA in young children.

Adolescent↗
PubMed1999

Neurite outgrowth in PC12 cells. Distinguishing the roles of ubiquitylation and ubiquitin-dependent proteolysis.

Nerve growth factor (NGF)-induced neurite outgrowth from rat PC12 cells was coincident with elevated (>/=2-fold) levels of endogenous ubiquitin (Ub) protein conjugates, elevated rates of formation of 125I-labeled Ub approximately E1 (Ub-activating enzyme) thiol esters and 125I-labeled Ub approximately E2 (Ub carrier protein) thiol esters in vitro, and enhanced capacity to synthesize 125I-labeled Ub-protein conjugates de novo. Activities of at least four E2s were increased in NGF-treated cells, including E2(14K), a component of the N-end rule pathway. Ubiquitylation of 125 I-labeled beta-lactoglobulin was up to 4-fold greater in supernatants from NGF-treated cells versus untreated cells and was selectively inhibited by the dipeptide Leu-Ala, an inhibitor of Ub isopeptide ligase (E3). However, Ub-dependent proteolysis of 125I-labeled beta-lactoglobulin was not increased in supernatants from NGF-treated cells, suggesting that neurite outgrowth is promoted by enhanced rates of synthesis (rather than degradation) of Ub-protein conjugates. Consistent with this observation, neurite outgrowth was induced by proteasome inhibitors (lactacystin and clasto-lactacystin beta-lactone) and was associated with elevated levels of ubiquitylated protein and stabilization of the Ub-dependent substrate, p53. Lactacystin-induced neurite outgrowth was blocked by the dipeptide Leu-Ala (2 mM) but not by His-Ala. These data 1) demonstrate that the enhanced pool of ubiquitylated protein observed during neuritogenesis in PC12 cells reflects coordinated up-regulation of Ub-conjugating activity, 2) suggest that Ub-dependent proteolysis is a negative regulator of neurite outgrowth in vitro, and 3) support a role for E2(14K)/E3-mediated protein ubiquitylation in PC12 cell neurite outgrowth.

Animals↗
PubMed2000

An investigation of the water-binding properties of protein + sugar systems.

The water-binding properties of sucrose + beta-lactoglobulin and trehalose + beta-lactoglobulin prepared by freeze-drying, spray-drying and evaporation from solution have been studied using gravimetric methods. The hydration characteristics of the individual sugars are dependent on the method of drying, and different isotherms have been recorded for each of the sample preparations. However, the initial hydration isotherms produced for the sugar + protein samples appear to be very similar for each sample type irrespective of drying method, with the sugar present in the amorphous glassy form in all cases. There is evidence of interaction between the sugar in this form and protein, specifically in the hydration region where single hydrogen-bonded water is expected to be bound. The magnitude of the interaction appears to be of the same order for both sugars. Irrespective of the method of preparation, rehydration of the sugar + protein complexes above a critical value causes a transition resulting in the sugar adopting a crystalline form and phase separation of the sugar and protein. In this crystalline form there is no evidence of interaction between the sugar and protein. For sugar + protein samples prepared by evaporation from solution, a small amount (approximately 3% by weight) of water is trapped in the complex even under extreme conditions of dehydration.

Animals↗
PubMed2001

A vegan diet free of gluten improves the signs and symptoms of rheumatoid arthritis: the effects on arthritis correlate with a reduction in antibodies to food antigens.

OBJECTIVE: Whether food intake can modify the course of rheumatoid arthritis (RA) is an issue of continued scientific and public interest. However, data from controlled clinical trials are sparse. We thus decided to study the clinical effects of a vegan diet free of gluten in RA and to quantify the levels of antibodies to key food antigens not present in the vegan diet. METHODS: Sixty-six patients with active RA were randomized to either a vegan diet free of gluten (38 patients) or a well-balanced non-vegan diet (28 patients) for 1 yr. All patients were instructed and followed-up in the same manner. They were analysed at baseline and after 3, 6 and 12 months, according to the response criteria of the American College of Rheumatology (ACR). Furthermore, levels of antibodies against gliadin and beta-lactoglobulin were assessed and radiographs of the hands and feet were performed. RESULTS: Twenty-two patients in the vegan group and 25 patients in the non-vegan diet group completed 9 months or more on the diet regimens. Of these diet completers, 40.5% (nine patients) in the vegan group fulfilled the ACR20 improvement criteria compared with 4% (one patient) in the non-vegan group. Corresponding figures for the intention to treat populations were 34.3 and 3.8%, respectively. The immunoglobulin G (IgG) antibody levels against gliadin and beta-lactoglobulin decreased in the responder subgroup in the vegan diet-treated patients, but not in the other analysed groups. No retardation of radiological destruction was apparent in any of the groups. CONCLUSION: The data provide evidence that dietary modification may be of clinical benefit for certain RA patients, and that this benefit may be related to a reduction in immunoreactivity to food antigens eliminated by the change in diet.

Adult↗
PubMed1995

Antibody titre and avidity in saliva and serum are not impaired in mildly to moderately undernourished children.

Avidities and titres of serum IgA, IgM, IgG, and salivary IgA antibodies for poliovirus type 3, beta-lactoglobulin and E. coli O antigens were analysed in well-nourished (n=58) and mildly to moderately undernourished (n = 18) 1-7-year-old children, living in slums in São Paulo, Brazil, with irregular water supply and no sewage system. The serum titres of antibodies in the undernourished children were not statistically different from the well-nourished ones, except for the IgA anti-beta-lactoglobulin titres which were lower in the youngest undernourished group. In respect to avidity of the serum antibodies, no statistical difference was observed, except that the youngest undernourished children presented avidities of serum IgA anti-E. coli O antigens higher than those of the well-nourished children of the same age range. With respect to salivary titres and avidities no difference was observed between well-nourished and undernourished children. The fact that the undernourished children were not deficient in antibody amount or quality, as measured in this study, suggests that in this respect their immune system was not impaired.

Antibodies, Bacterial↗
PubMed1983

Partial hydrolysis of cow's milk proteins by human trypsins and elastases in vitro.

The hydrolysis of bovine alpha-lactalbumin, beta-lactoglobulin, and casein by human anodal and cathodal trypsins and elastases was studied with the aid of electroimmunoassay and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The rate of hydrolysis of the various proteins by cathodal elastase exceeded that by anodal or cathodal trypsin and anodal elastase. Casein was hydrolyzed more efficiently than alpha-lactalbumin or beta-lactoglobulin. The hydrolysis of the three proteins occurred at a considerably slower rate when present in crude form, as in cow's milk, than when in purified form.

Animals↗
PubMed1996

Effects of native and hydrolyzed whey protein on intestinal repair of severely starved rats at weaning.

The aim of this study was to evaluate the effect of two sources of dietary nitrogen (isolated whey protein and hydrolyzed whey protein) on the intestinal repair of malnourished rats at weaning. The malnutrition was achieved by a 3 days' starvation period. Normally fed male Wistar rats were used as controls. Intestinal repair was studied after a refeeding period of 4 days. The parameters studied included nitrogen balance, lactase, sucrase, isomaltase, and maltase activities of the jejunum; liver acetylcholinesterase and glutamate dehydrogenase activities; and the serum amino acid profile. In addition, tests of intestinal permeability to macromolecules were performed by measurement of ovalbumin and beta-lactoglobulin in serum. Both diets of led to the recovery of the severely starved rats, in terms of the values of all the parameters evaluated. The serum beta-lactoglobulin was the only exception, because its concentration was significantly lower in the normally fed animals. This study suggests that the intestinal mucosal barrier is not completely repaired, even after a 4-day refeeding period, to the point of being suitable to accept an increase in the uptake of antigens.

Acetylcholinesterase↗
PubMed1994

Protective action of diosmectite treatment on digestive disturbances induced by intestinal anaphylaxis in the guinea-pig.

METHODS: Colonic transit time, faecal moisture and intestinal permeability were assessed in guinea-pigs sensitized intraperitoneally with cow's milk and challenged with an oral administration of beta-lactoglobulin. One group of animals was treated for 1 week with diosmectite (500 mg.kg/day) and another with placebo. A control group was not sensitized but treated with diosmectite. RESULTS: In sensitized animals receiving placebo, challenge with beta-lactoglobulin induced a significant (P < 0.05) decrease in colonic transit time, and increases in faecal moisture and intestinal permeability. These changes were not observed in animals treated with diosmectite. CONCLUSION: Diosmectite pre-treatment protects against allergic digestive disturbances induced by antigen administration in guinea-pigs sensitized to cow's milk.

Anaphylaxis↗
PubMed1994

Anaphylactic colonic hypersecretion in cow's milk sensitized guinea-pigs depends upon release of interleukin-1, prostaglandins and mast cell degranulation.

METHODS: The effect of beta-lactoglobulin (beta-LGI) challenge on net water movements into the proximal colon and the role of Interleukin-1 (IL-1), prostaglandins and mast cell degranulation on the challenge-induced net water changes were assessed in vivo using isolated colonic loops in anaesthetized guinea-pigs immunized to bovine milk. RESULTS: beta-lactoglobulin challenge infused into the colonic loop during 30 min reversed the net water flux into a net secretion during the period of antigen infusion. Doxantrazole, a mast cell stabilizing agent, administered 120 min before challenge infusion, suppressed challenge-induced hypersecretion. Similarly recombinant IL-1 receptor antagonist protein abolished the antigen-induced colonic secretory effect. Indomethacin, a prostaglandin synthesis inhibitor, administered 20 min prior to antigen infusion, significantly (P < 0.05) reduced, but did not abolish, the challenge-induced colonic secretory effect. CONCLUSIONS: These results suggest that IL-1 plays an important role in antigen challenge-induced colonic hypersecretion which involves mast cell degranulation and prostaglandin release.

Anaphylaxis↗
PubMed1990

Influence of the G2m(n) allotype and age on IgG subclass distribution in antibodies to dietary proteins in children with coeliac disease.

In 47 children with coeliac disease, IgG1 was the dominant IgG subclass (mean 61%) in antibodies to gliadin, a protein component of wheat; IgG2 (22%) and IgG3 (15%) were also found. Very little IgG4 (3%) was detected. Both G2m(n)allotype and the age of the patient had an independent effect on the subclass distribution. The effects of these two factors seem similar to those described earlier on responses to polysaccharides; both G2m(n) and age increase the expression of IgG2 antibodies and concomitantly the share of IgG1 is decreased. The average increase in the share of IgG2 brought about by n/n-genotype was 25.3% (95% confidence limits 12.2-38.4%; P less than 0.001) and that brought about by each additional year of age of the patient 1.1% (0.3-1.9%; P = 0.006). IgG antibodies to beta-lactoglobulin and ovalbumin consisted mainly of IgG1 and IgG4. The titres of the IgG subclasses were low and in many cases below detection level. No association with G2m(n) genotype and share of IgG1 or IgG4 could be demonstrated. Age was inversely correlated with the shares of IgG1 in antibodies to beta-lactoglobulin and ovalbumin.

Adolescent↗
PubMed1981

Differences in specific antibody responses of human tonsillar cells to an oral and a parenteral antigen.

Tonsillar lymphocytes stimulated in vitro with either beta-lactoglobulin or with tetanus toxoid were shown to produce specific antibodies by a direct plaque assay and by radioimmunoprecipitation of the culture supernatants. There was a sixfold increase in the number of IgA-secreting cells in response to beta-lactoglobulin; no such effect was seen in response to tetanus toxoid, where a fivefold rise in IgG-secreting cells occurred. These differences in antibody response are probably due to the route of initial antigen presentation. Those antigens priming the mucosa-associated lymphoid system stimulate mainly IgA-producing cells, in contrast to parenteral antigens, which elicit a predominantly IgM and IgG response.

Administration, Oral↗
PubMed1992

Aberrations in titre and avidity of serum IgM and IgG antibodies to microbial and food antigens in IgA deficiency.

The antibody levels and relative avidity of serum IgM and IgG antibodies against E. coli O antigens, poliovirus type 1 and beta-lactoglobulin were determined with enzyme-linked immunosorbent techniques in IgA deficient (IgAd) patients with frequent respiratory tract infections and healthy IgAd individuals. Healthy individuals with normal immunoglobulin levels served as controls. The IgM antibody levels against the bacterial, viral and food antigens and the IgG antibody levels against the bacterial antigens were significantly higher in the IgAd group with recurrent infections than in the group of healthy IgAd individuals. The symptomatic IgAd group had significantly higher levels of the IgG antibodies against the bacterial antigen, also when compared with controls. In contrast the healthy IgAd individuals had the highest avidities of IgM antibodies to the viral and food antigens. The high avidities of antibodies could be a compensatory host defence mechanism in IgAd. These aberrations may appear as a consequence of increased mucosal exposure in IgAd to antigens such as E. coli or beta-lactoglobulin, but presumably not to poliovirus which is only exceptionally present in the milieux. They could also be a result of the previously suggested dysregulation of antibody responses in IgAd.

Antibody Affinity↗
PubMed1996

Identification of casein as the major allergenic and antigenic protein of cow's milk.

The objective of this study was to analyze both the allergenicity and immunogenicity of cow's milk proteins. To this end, 80 milk-atopic patients were selected on the basis of the presence of cow's milk-specific IgE antibodies in serum and compatible clinical history. Fifteen patients allergic to other allergens and 10 nonatopic subjects were studied as controls. The specificity of serum IgG and IgE antibodies was determined by immunoblotting, employing both cow's milk and milk components, i.e., alpha- and beta-casein, beta-lactoglobulin, and alpha-lactalbumin separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The experiments showed that casein-specific IgE antibodies were present in all (80/80) sera examined; 10/80 showed reactivity to beta-lactoglobulin, and 5/80 showed reactivity to alpha-lactalbumin. None of the 25 negative control sera analyzed showed the presence of specific IgE antibodies against milk proteins. These results were similar to those corresponding to the detection, by the radioallergosorbent test, of IgE antibodies against the milk components coupled to paper disks. All sera from milk-atopic patients also showed IgE reactivity against a high-molecular-mass fraction that hardly enters the gel. This fraction, after separation by gel filtration and treatment with beta-mercaptoethanol and urea, was shown by SDS-PAGE analysis to be formed by casein monomers. All sera analyzed by immunoblotting reacted against the components corresponding to casein monomers. Inhibition of immunoblotting by adsorption with different milk components confirmed that those high-molecular-mass aggregates are formed by casein components. The results presented here strongly suggest that casein is the major allergenic component of cow's milk.

Adolescent↗
PubMed1996

Fetal peripheral blood mononuclear cell proliferative responses to mitogenic and allergenic stimuli during gestation.

Blood samples were obtained from fetuses and premature babies (n = 51) (15-34 weeks gestation) to determine at what stage the fetal immune system was able to produce a positive proliferative response to common allergens. Peripheral blood mononuclear cells (PBMC) were stimulated with the mitogen, phytohaemagglutinin (PHA), and the allergens, house dust mite, cat fur, birch tree pollen, beta-lactoglobulin, ovalbumin and bee venom (mellitin). Results were expressed as ratios of stimulated to unstimulated 3H thymidine incorporation, and as percent positive responders. There was an increase in proliferation ratio which correlated with increasing gestational age for PHA (p < 0.0001), cat fur (p = 0.042), birch pollen (p = 0.022) and beta-lactoglobulin (p = 0.006). The point in gestation when cells from some individuals began responding to the allergens with a ratio of 2.0 was at approximately 22 weeks. PBMC proliferative response ratios were higher from samples from babies > 22 weeks gestation compared to < 22 weeks for the mitogen and all allergens, except mellitin. There was also a greater proportion of positive responders from samples > 22 weeks compared to < 22 weeks for the mitogen and all allergens, except mellitin. Maternal exposure to birch pollen, which has a discrete season, was assessed to determine whether exposure had occurred at 22 weeks gestation or beyond. Results showed a higher proliferative response in infant cells stimulated with birch pollen (p = 0.005) and higher proportion of positive responders (p = 0.01) in the group of babies whose mothers had been exposed to birch pollen beyond 22 weeks, compared to those whose mothers had not been so exposed. These results suggest that in utero fetal exposure to an allergen from around 22 weeks gestation may result in primary sensitisation to that allergen, leading to positive proliferative responses, at birth.

Adult↗
PubMed1991

Complete sequence and model for the A2 subunit of the carotenoid pigment complex, crustacyanin.

The complete sequence has been determined for the A2 subunit of crustacyanin, an astaxanthin-binding protein from the carapace of the lobster Homarus gammarus. The polypeptide chain is 174 residues long and is similar to proteins of the retinol-binding protein superfamily. Some regions of the sequence are most similar to the retinol-binding protein, beta-lactoglobulin subgroup, while the disulphide bonding pattern is more akin to that seen in the porphyrin binding proteins insecticyanin and bilin-binding protein. It is beginning to appear as though this superfamily of proteins, characterized by a similar gross structural framework, may be further subdivided into interrelated subclasses. Model building based on the coordinates of the known structure of human plasma retinol-binding protein and on empirical prediction algorithms has allowed the putative identification of side-chains which line the binding cavity. This pocket is larger than in retinol binding protein and beta-lactoglobulin but does not allow the carotenoid to adopt a folded conformation. The amino acid composition of the pocket does not support a 'charge-shift'-type hypothesis to support the bathochromic shift phenomenon which takes place on interaction of the chromophore with the protein. Instead aromatic side-chains may play a prominent role.

Amino Acid Sequence↗