Beta Lg Research Articles

Protein−Lipid Interactions at the Air−Water Interface

Protein-lipid interactions play an important role in a variety of fields, for example in pharmaceutical research, biosensing, or food science. However, the underlying fundamental processes that govern the interplay of lipids and proteins are often very complex and are therefore studied using model systems. Here, Langmuir monolayers were used to probe the interaction of a model protein with lipid films at the air-water interface. The protein beta-lactoglobulin (beta lg) is the major component in bovine milk serum, where it coexists with the milk fat globular membrane. During homogenization of milk, beta lg adsorbs to the interface of lipid fat globules and stabilizes the oil-in-water emulsion. pH and ionic strength of the subphase had a significant effect on the surface activity of the protein. Additionally, by using lipids with different charges, it could be shown that the interactions between beta lg and a phospholipid layer were driven by hydrophobic as well as by electrostatic interactions. beta lg preferentially interacted with phospholipids in an unfolded state. This could be either achieved by denaturation at the air-water interface or due to electrostatic interactions that weaken the intramolecular forces of the protein.

Detection of pH 4.6 Insoluble β-Lactoglobulin in Heat-Treated Milk and Mozzarella Cheese

Different protein aggregates including beta-lactoglobulin (beta lg) were detected in the pH 4.6 insoluble fraction recovered from actual heat-treated milk samples by gel electrophoresis and immunoblotting. A competitive enzyme-linked immunosorbent assay (ELISA) using anti-beta lg polyclonal antibodies was developed to analyze the beta lg partition in the protein fractions obtained upon acidification of both milk and Mozzarella cheese at pH 4.6. According to ELISA determinations, nearly 90% of the pH 4.6 soluble beta lg included in raw milk was found in the pH 4.6 insoluble fraction of ultrahigh temperature (UHT)-treated milk. As concerns Mozzarella cheese analysis, ELISA results indicated that about 36% of the total beta lg milk content was transferred from pasteurized milk to Mozzarella cheese, whereas less than 0.5% was transferred from raw milk. The pH 4.6 insoluble beta lg proved to be a suitable indicator of the intensity of the heat treatment applied to milk. The ELISA-based detection of this parameter was suggested for quality control of both drinking milk and raw milk cheese.

A Simple DNase Model System Comprising a Dinuclear Zn(II) Complex in Methanol Accelerates the Cleavage of a Series of Methyl Aryl Phosphate Diesters by 1011−1013

The di-Zn(II) complex of 1,3-bis[ N1, N1'-(1,5,9-triazacyclododecyl)]propane with an associated methoxide ( 3:Zn(II) 2: (-)OCH 3) was prepared and its catalysis of the methanolysis of a series of fourteen methyl aryl phosphate diesters ( 6) was studied at s (s)pH 9.8 in methanol at 25.0 +/- 0.1 degrees C. Plots of k obs vs [ 3:Zn(II) 2: (-)OCH 3] free for all members of 6 show saturation behavior from which K(M) and kcat (max) were determined. The second order rate constants for the catalyzed reactions (kcat (max)/K(M)) for each substrate are larger than the corresponding methoxide catalyzed reaction (k 2 (-OMe)) by 1.4 x 10(8) to 3 x 10 (9)-fold. The values of k cat (max) for all members of 6 are between 4 x 10(11) and 3 x 10(13) times larger than the solution reaction at s (s)pH 9.8, with the largest accelerations being given for substrates where the departing aryloxy unit contains ortho-NO 2 or C(O)OCH 3 groups. Based on the linear Brønsted plots of k cat (max) vs s (s)pKa of the phenol, beta lg values of -0.57 and -0.34 are determined respectively for the catalyzed methanolysis of "regular" substrates that do not contain the ortho-NO 2 or C(O)OCH 3 groups, and those substrates that do. The data are consistent with a two step mechanism for the catalyzed reaction with rate limiting formation of a catalyst-coordinated phosphorane intermediate, followed by fast loss of the aryloxy leaving group. A detailed energetics calculation indicates that the catalyst binds the transition state comprising [CH 3O (-): 6], giving a hypothetical [ 3:Zn(II) 2:CH 3O (-): 6] complex, by -21.4 to -24.5 kcal/mol, with the strongest binding being for those substrates having the ortho-NO 2 or C(O)OCH 3 groups.

Energetics of protein homodimerization: Effects of water sequestering on the formation of β‐lactoglobulin dimer

Transient protein-protein interactions are functionally relevant as a control mechanism in a variety of biological processes. Analysis of the 3D structure of protein-protein complexes indicates that water molecules trapped at the interface are very common; however, their role in the stability and specificity of protein homodimer interactions has been not addressed yet. To provide new insights into the energetic bases that govern the formation of highly hydrated interfaces, the dissociation process of bovine beta lg variant A at a neutral pH was characterized here thermodynamically by conducting dilution experiments with an isothermal titration calorimeter. Association was enthalpically driven throughout the temperature range spanned. DeltaH and deltaC(p) were significantly more negative than estimates based on surface area changes, suggesting the occurrence of effects additional to the dehydration of the contact surfaces between subunits. Near-UV CD spectra proved to be independent of protein concentration, indicating a rigid body-like association. Furthermore, the process proved not to be coupled to significant changes in the protonation state of ionizable groups or counterion exchange. In contrast, both osmotic stress experiments and a computational analysis of the dimer's 3D structure indicated that a large number of water molecules are incorporated into the interface upon association. Numerical estimates considering the contributions of interface area desolvation and water immobilization accounted satisfactorily for the experimental deltaC(p). Thus, our study highlights the importance of explicitly considering the effects of water sequestering to perform a proper quantitative analysis of the formation of homodimers with highly hydrated interfaces.

A High-Throughput Method for Detection of Protein Self-Association and Second Virial Coefficient Using Size-Exclusion Chromatography Through Simultaneous Measurement of Concentration and Scattered Light Intensity

To characterize protein self-association along with second virial coefficient (a measure of solution nonideality) using size-exclusion chromatography (SEC) utilizing a novel flow cell that is capable of simultaneously measuring protein concentration and scattered light intensity. beta-lactoglobulin A (beta Lg), known to exhibit NaCl-dependent monomer-dimer equilibrium at pH 3.0, was used as the model protein. A range of concentrations and corresponding scattered light intensities, obtained in the eluting peak from a single protein injection, in different solution conditions, were used to generate the Debye plots [Formula: see text]. The Debye light scattering equation was modified to include the monomer-dimer equilibrium model and the second virial coefficient to analyze the data obtained. Debye plots of beta Lg, while linear at pH 2.3, 0 M NaCl (pure monomer) and at pH 3.0, 1 M NaCl (pure dimer), showed curvature at pH 3.0, for varying NaCl concentrations (0.02-0.5 M). The curvature was indicative of the association behavior of this protein. The modified Debye light scattering equation, when fit onto the nonlinear Debye plots, yielded apparent K (a) values ranging from 10(2) to 10(5) M(-1) under various solution conditions. The apparent K (a) values obtained from this method followed similar trend to those reported in literature. SEC combined with simultaneous detection of scattered light intensity and concentration provides a rapid means of detection of protein self-association. The short duration of sample detection and analysis combined with SEC makes this method a useful tool for high-throughput characterization of protein association during early stages of protein formulation.

Ketene-forming eliminations from aryl bis(4'-chlorophenyl)acetates promoted by R2NH-R2NH2+ in aqueous MeCN. Change of mechanism.

Elimination reactions of (4'-ClC6H4)2CHCO2C6H3-2-X-4-NO2 promoted by R2NH-R2NH2+ in 70 mol% MeCN (aq.) have been studied kinetically. The reactions are second-order and exhibit Brönsted beta = 0.44-0.86 and /beta(lg)/ = 0.41-0.71. The Brönsted beta decreased with a poorer leaving group and /beta(lg)/ increased with a weaker base. The results are consistent with an E2 mechanism. When X=H, the reaction proceeded by the concurrent E2 and Elcb mechanism.

Reversible conformational change in beta-lactoglobulin A modified with N-ethylmaleimide and resistance to molecular aggregation on heating.

beta-Lactoglobulin A (beta LG A) modified with N-ethylmaleimide (NEM-beta LG A) was purified by ion exchange chromatography, and modification of beta LG A by NEM was confirmed by time of flight mass spectrometry and 5,5'-dithiobis(2-nitrobenzoic acid) methods. The fluorescent spectrum of NEM-beta LG A was slightly different from that of native beta LG A. NEM-beta LG A gave no polymerization after heating at 80 degrees C and pH 7.5, as shown by polyacrylamide gel electrophoresis. Conformational change of NEM-beta LG A was observed at 80 degrees C by ultraviolet differential spectra, whereas after cooling it recovered to its original state as before heating, indicating apparent reversible thermal denaturation. Native beta LG A is resistant to pepsin hydrolysis, whereas heated beta LG A was easily hydrolyzed by pepsin. NEM-beta LG A before heating was also resistant to pepsin hydrolysis, and after heating NEM-beta LG A was still resistant to pepsin hydrolysis. These results indicate that NEM-beta LG A maintained a conformation similar to its native form even after heating. Addition of 0.2 M NaCl to the beta LG A heated under salt-free condition induced polymerization of heated beta LG A molecules, but not that of heated NEM-beta LG A. This seemed to indicate that the formation of inter- or intramolecular disulfide linkage made the heat-induced conformational change of beta LG A irreversible.

Characterization of baboon (Papio hamadryas) milk proteins.

The major proteins of baboon milk were identified as beta-lactoglobulin (beta LG), alpha-lactalbumin (alpha LA), lysozyme, lactoferrin, casein, and albumin by immobiline isoelectric focusing, SDS-PAGE, immunoblotting of gels with rabbit antisera to human alpha LA, lysozyme, and albumin and bovine beta LG and casein, and N-terminal sequencing of proteins blotted from gels. The first 30 N-terminal residues of baboon beta LG are identical to those of macaque (Macaca fasicularis) beta LG except for a (D/N) polymorphism at residue 2. The complete cDNA sequence and derived amino acid composition of beta LG were elucidated using RT-PCR amplification of poly(A)+ mRNA purified from lactating mammary gland. Baboon beta LG consists of 168 amino acid residues (M(r) 20,750) and is the longest beta LG identified to date. beta LG and alpha LA polymorphisms with three (A, B, and C) and two (A and B) variants, respectively, were detected by immobiline IEF, pH 4-6, of individual baboon milk samples at varying stages of lactation.

Nucleosome organisation of the beta-lactoglobulin gene. Transcription complex formation.

In most mammals, the major whey protein beta-lactoglobulin (beta lg) represents a marker for tissue-specific, temporally regulated gene expression in the mammary gland. Prolactin, the major lactogenic stimulus which activates beta lg expression acts through a cytoplasmic signalling cascade ending in the activation of the transcription factor STAT5. Although much is known about the complexities of this signalling cascade, little is known about how this transcription factor functions within the context of chromatin. Using DNaseI as a probe of chromatin structure we have identified temporally regulated elements within the ovine beta lg gene domain. The appearance of these hypersensitive sites accompanies changes in expression state of the beta lg gene. Changes in DNaseI hypersensitivity at the proximal promoter region, while reflecting STAT5 activation, is not dependent upon STAT5 interaction at this site. We have mapped the nucleosome positions over the entire beta lg gene, both in vitro using the monomer extension assay and in vivo using cuprous phenanthroline to probe for nucleosome-linker positions. The specific positioning pattern detected, which reflects strong sequence-directed positioning over the proximal promoter, complement the STAT5 consensus sites within this region. The comparison of both the functional and chromatin data enables a model for beta lg gene transcription to be developed.

Molecular cloning and sequence analysis of the cDNA encoding beta-lactoglobulin in Bubalus bubalis.

The cDNA for bubaline beta-lactoglobulin (beta lg) has been cloned through RT-PCR approach and sequenced. Sequence data showed a single open reading frame coding for a protein of 180 amino acids with a signal sequence of 18 amino acid residues. Comparison with other ruminant beta lg sequences revealed a high homology indicating the protein to be conserved through evolution. The degree of homology, at amino acid level, is 96.1%, 95.6%, 93.9% and 63.7% with goat, sheep, cow and pig, respectively.

Metal-substrate interactions facilitate the catalytic activity of the bacterial phosphotriesterase.

The bacterial phosphotriesterase from Pseudomonas diminuta is a zinc metalloenzyme which catalyzes the hydrolysis of a variety of organophosphorus nerve agents with high efficiency. The active site of the enzyme consists of a coupled binuclear metal center embedded within a cluster of histidine residues. Potential protein-substrate interactions at the active site were probed by a systematic variation of metal identity, leaving group potential, phosphate host, and amino acid replacement. In order to determine the roles of these metal ions in binding and catalysis, the microscopic rate constants and kinetic parameters were obtained with various divalent cations. The divalent cations that were utilized in this investigation consisted of Co2+, Ni2+, Cd2+, Zn2+, Mn2+, and the mixed-metal Zn2+/Cd2+ hybrid. The leaving group potential and phosphate host were varied by altering the pKa of the departing substituted phenol or thiophenol in either a diethyl phosphate or a diethyl thiophosphate substrate. The Brønsted plots for the nonenzymatic hydroxide catalyzed hydrolysis of these substrates showed a linear dependence between the pseudo-first-order rate constant and the pKa of the leaving group. Enzymatic activities of the wild-type enzyme with these same substrates varied by over 7 orders of magnitude over the entire experimental pKa range (4.1-10.3), and the corresponding Brønsted plots were nonlinear. Those substrates with leaving groups with high pKa values were limited by the rate of bond cleavage while those substrates having leaving groups with low pKa values were limited by a conformational change or binding event. Thiophosphate substrates having leaving groups with high pKa values were better substrates than the corresponding phosphate analogues. These results are consistent with the direct coordination of one or both metal ions with the phosphoryl sulfur or oxygen atom of the substrate. A large dependence of the rate on the leaving group rules out the possibility of protonation of the leaving group or electrostatic interaction of the leaving group oxygen (or sulfur) with a metal ion or cationic group at the active site. The large differences in the size of the beta lg over the range of metal ions utilized by the enzyme indicate that the metal ions polarize the phosphoryl group and alter the structure of the transition state. The values of V/K(m) for the enzyme-catalyzed hydrolysis for a series of substituted thiophenol analogues were 10(2)-10(3)-fold smaller than those obtained for the hydrolysis of the corresponding phenolic substrates, suggesting that the bulkier sulfur substituent in the leaving group may induce conformational restrictions at the active site. With the zinc-substituted H201N mutant enzyme, there was a large decrease in the rate of phosphotriester hydrolysis but essentially no change in the rate of thiophosphotriester hydrolysis relative to the values observed for the zinc-substituted wild-type enzyme. These results suggest that a direct perturbation in the ligand structure of the binuclear metal center induces alterations in the mechanism of substrate hydrolysis.

Relative Allergenicity of Cow’s Milk and Cow’s Milk-Based Formulas in an Animal Model

Human infants fed cow's milk or cow's milk-based formula may become intolerant to milk proteins partially because of allergic reactions. However, the relative allergenicity of these diets has not been clearly determined at the intestinal level. In this study, the allergenicity of cow's milk and milk-based formula was evaluated by examining intestinal anaphylaxis caused by challenge with a milk protein fraction, beta-lactoglobulin (beta LG), in guinea pigs orally sensitized to these diets. Colonic segments were removed and challenged with beta LG. An antigen-induced, anaphylactically mediated elevation of transmural short circuit current, which is caused by net chloride (Cl-) secretion, was measured in Ussing chambers as an index of local immune responsiveness. After removal of the colon, all guinea pigs were challenged by intracardiac injection with beta LG to examine the onset of bronchospasm, a test for systemic anaphylaxis. Whereas colonic segments from all guinea pigs fed whole cow's milk responded to challenge with beta LG, segments from only 60% of animals fed cow's milk formula responded to the challenge. In addition, the responders in the latter group had a significantly lower magnitude of beta LG-induced Cl- secretion compared with animals sensitized to whole cow's milk. In parallel, bronchospasm developed in all guinea pigs fed whole cow's milk. In the group of animals fed cow's milk formula, bronchospasm developed only in those who responded to the intestinal challenge. On the basis of these results, cow's milk-based formula has less sensitizing power than whole cow's milk in our animal model, and our approach is effective in testing allergenicity at the intestinal level.

Raising the pH of the pepsin‐catalysed hydrolysis of bovine whey proteins increases the antigenicity of the hydrolysates

Hypersensitivity to cow milk protein is frequently observed in infancy. Since the pH in the infant's stomach is relatively high (pH 3-4) compared with adults (pH 2), an incomplete digestion of the milk proteins is expected to occur. The determination of the degree of hydrolysis by pepsin of the four main proteins of bovine whey, i.e. alpha-lactalbumin (alpha La), beta-lactoglobulin (beta Lg), bovine serum albumin (BSA) and bovine immunoglobulin G (B-IgG), in the pH range 2.0-4.0 and of the antigenic properties of the resulting hydrolysates. Whey proteins were successively hydrolysed with pepsin at pH values ranging from 2.0 to 4.0 and with pancreatic enzymes at pH 7.5 using a pH-stat. The resulting hydrolysates were characterized by their degree of hydrolysis, and analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, gel permeation chromatography and immunologically by competitive enzyme-linked immunosorbent assay. In general, the degree of hydrolysis, the gel electrophoretic patterns, the contents of peptides of molecular mass > 5 kDa and the residual human-immunoglobulin E and G antigenicities of the hydrolysates did not differ much whether the pepsin incubation was done at pH 2.0 or 3.0. Pepsin incubation at pH 4.0, however, resulted in a decreased hydrolysis and enhanced residual antigenicity of alpha La, BSA and B-IgG, but not of beta Lg. The poor and slow degradation of the antigenic epitopes of whey proteins when pepsin digestion occurs under conditions that prevail in the stomach of infants could be of much importance for the development of cow milk hypersensitivity. The immature gastrointestinal mucosal barrier of infants allows large antigenic fragments of these proteins to pass into the systemic circulation.

Dependence of the phosphorylation of alkaline phosphatase by phosphate monoesters on the pKa of the leaving group

The hydrolysis and transphosphorylation reactions of a series of phosphate monoesters, ROPO3(2)-(R = 2,4-dinitrophenyl, 4-nitrophenyl, phenyl, glucose-1, glycerol-1, methyl, ethyl, and dodecyl), catalyzed by Escherichia coli alkaline phosphatase and a mutant enzyme, Ser102Cys, have been studied at alkaline pH using the rates of change in the 31P NMR signals of substrate, the hydrolysis product (inorganic phosphate), and the transphosphorylation product (O-Tris phosphate) as the assay. The kcat at pH 8.0 for the wild-type enzyme is approximately 30 s-1 and is independent of the nature of the R group, when the pKa of the leaving group is < 10. Under these conditions the rate of phosphorylation is much faster than dissociation of inorganic phosphate, 15-60 s-1. If the pKa of the leaving group is between 10 and 15, phosphorylation and dissociation of the product phosphate both contribute to the rate limit. If the pKa of the leaving group is > 15, phosphorylation is rate limiting. A Bronsted plot of log kcat vs pKa of the leaving group for those substrates for which phosphorylation is rate limiting yields a beta lg of approximately -0.6. In contrast to the wild-type enzyme, the log kcat values for the S102C mutant enzyme catalyzing the hydrolysis of phosphate esters are linearly dependent on the pKa's of the leaving group throughout the range of pKa from 4 to 16. Phosphorylation of C102 is the rate controlling step, and kcat is independent of the Tris concentration as predicted for rate limiting phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)

Nature of the rate-determining steps of the reaction catalyzed by the Yersinia protein-tyrosine phosphatase.

Product inhibition and 18O exchange experiments suggest that the Yersinia protein-tyrosine phosphatase-catalyzed phosphate monoester hydrolysis proceeds through at least two different chemical steps, i.e. the formation and breakdown of a covalent phosphoenzyme intermediate. The pH dependence of kcat values is bell-shaped, with the apparent pKa derived from the acidic limb of the profile at 4.6 for both p-nitrophenyl phosphate and beta-naphthyl phosphate, whereas the apparent pKa derived from the basic limb of the profile is substrate-dependent, with apparent pKa values of 5.2 and 5.8 for p-nitrophenyl phosphate and beta-naphthyl phosphate, respectively. Twelve aryl phosphates with leaving groups having pKa values from approximately 7 to 10 are also examined as substrates at two pH values. At pH 4.0, the beta lg values is effectively zero, whereas at pH 7.5, a beta lg value of 0.16 is observed. Collectively, our results suggest that the rate-determining step under acidic conditions corresponds to the breakdown of the phosphoenzyme intermediate, whereas under more alkaline conditions, substrate effects also contribute to the rate-limiting step. A model is proposed for the mechanism of the Yersinia protein-tyrosine phosphatase-catalyzed reaction.

Intestinal anaphylaxis: rapid changes in mucosal ion transport and morphology.

Anaphylactic reactions can be elicited by beta-lactoglobulin (beta LG) in the isolated colonic and ileal mucosae of guinea pigs fed cow milk. We have shown that these tissues respond to antigen challenge with a rapid rise in short-circuit current (SCC) due to net chloride secretion in the colon and less well-characterized ion movements in the ileum. Here, tissues were characterized morphologically at or near the peak of the SCC response 2 min after antigen addition. These sensitized preparations, or naive tissues passively sensitized with immune sera and challenged with beta LG, showed numerous structural alterations that correlated significantly with the change in SCC. In the ileum, these changes included compound exocytosis of mucous granules from goblet cells of crypts but not of villi and a decrease in the intercellular space of the crypts. In contrast, colonic goblet cells of both crypts and luminal surface showed no evidence of secretion, but crypt architecture was reproducibly and characteristically altered. Thus antigen challenge of intestinal mucosa results in temporally related but diverse changes in morphology and ion transport.

Purification and mechanistic properties of an extracellular alpha-L-arabinofuranosidase from Monilinia fructigena.

1. The alpha-L-arabinofuranosidase isoenzyme designated AFIII [Laborda, Archer, Fielding & Byrde (1974) J. Gen. Microbiol. 81, 151-163] was purified by sequential isoelectric focusing, hydrophobic chromatography, gel filtration and chromatofocusing. 2. The enzyme is a monomer of Mr 40,000. 3. On inactivation of the enzyme with 3H-labelled 1-alpha-L-arabinofuranosylmethyl-3-p-nitrophenyltriazene, 0.64 mol of alpha-L-arabinofuranosylmethyl residues/mol of enzyme is estimated to become attached to protein. 5. Neither first-order nor second-order rate constants for hydrolyses of aryl alpha-L-arabinofuranosides are dependent upon leaving-group acidity [beta lg(V) = -0.16 +/- 0.11; Beta lg(V/K) = -0.11 +/- 0.07; n = 7; delta pKa = 4.5] 6. Bond-breaking is nonetheless rate-limiting, as is shown by a value of 18(V) of 1.030 +/- 0.007 for the hydrolysis of p-nitrophenyl arabinoside. 7. Proton-donation to the leaving group is thus far advanced at the rate-limiting transition state for this enzyme. 8. Four alpha-L-arabinofuranosyl pyridinium salts are substrates, and an approximate beta lg(V) value of -0.9 can be estimated. 9. The absolute rate enhancement with the 4-bromoisoquinolinium salt, 2.5 X 10(9), is comparable with that observed with pyranosidases. 10. Ring-opening mechanisms can therefore be dismissed, even though they are known in the acid-catalysed hydrolysis of arabinofuranosides.

Antibody production to milk proteins in the jejunal mucosa of children with Cow's milk protein intolerance.

A direct immunofluorescent technique with fluorescein-labeled antigens was used to examine the jejunal mucosa for specific antibody reduction in 23 children aged 2--25 months referred for diagnostic biopsy. Plasma cells containing antibodies to beta-lactoglobulin (beta LG) or bovine serum albumin (BSA) were present in 11 of 16 biopsies from patients with cow's milk protein intolerance (CMPI) but only one of eight controls, all on a diet containing cow's milk. Antibody-containing cells (ACC) constituted approximately 0.5--5% of the total (IgA + IgM) plasma cell population. Their presence did not correlate with either histologic abnormality or IgA plasma cell count. Double immunofluorescence with rhodamine-labeled antigens and fluorescein-labeled class-specific anti-human immunoglobulin sera showed the ACC to be approximately 70% IgA cells and 30% IgE cells.

Immediate hypersensitivity reaction to beta-lactoglobulin in the epithelium lining the colon of guinea pigs fed cows' milk.

Isolated epithelia from the colons of guinea pigs fed cows' milk have been studied in vitro under short circuit current conditions. When challenged with beta-lactoglobulin (beta LG) from the basolateral side, a large, transient inward flowing current was recorded. Pharmacological and ion flux measurements indicated this was due to inappropriate chloride secretion. There was no effect of beta LG on normal epithelia. The effects of challenge with beta LG were abolished by pretreatment with indomethacin, a prostaglandin synthetase inhibitor. By exposing epithelia from water-drinking animals in vitro to serum from milk-drinking guinea pigs sensitivity to beta LG could be conferred.