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Influence of cow's milk proteins and gluten on human duodenal mucosa in organ culture.

Forty-five duodenal biopsies from 33 children and 3 adult patients were maintained in organ culture for 24 h and exposed to various cow's milk proteins and gluten. In 10 of 11 celiac patients with a flat duodenal mucosa, and in 2 of 4 patients with partial villous atrophy, a significant reduction in the mean enterocyte height was found after in vitro gluten exposure, compared to culture in basic culture medium. Three patients had coexisting celiac disease and cow's milk protein intolerance. alpha-Lactalbumin and beta-lactoglobulin exhibited toxic effects on flat biopsies from two of these patients, and casein was toxic in one. In 10 patients with cow's milk protein intolerance, a significant reduction in enterocyte height was noted in one case with gluten, and in three patients with casein and lactoglobulin, whereas lactalbumin did not affect the tissues. In seven control patients having a normal duodenal mucosa, no in vitro influences were noted, whereas in four patients with partial villous atrophy, a toxic reaction to gluten was seen in one and a reduced enterocyte height was seen after lactoglobulin exposure in another. In vitro toxicity induced by gluten corresponded well with the diagnosis of celiac disease, whereas toxic reactions to cow's milk proteins during organ culture were inconsistent in cow's milk intolerance, except for cases in which a marked enteropathy was documented.

Adolescent↗

Transgenic mice secreting coronavirus neutralizing antibodies into the milk.

Ten lines of transgenic mice secreting transmissible gastroenteritis coronavirus (TGEV) neutralizing recombinant monoclonal antibodies (rMAbs) into the milk were generated. The rMAb light- and heavy-chain genes were assembled by fusing the genes encoding the variable modules of the murine MAb 6A.C3, which binds an interspecies conserved coronavirus epitope essential for virus infectivity, and a constant module from a porcine myeloma with the immunoglobulin A (IgA) isotype. The chimeric antibody led to dimer formation in the presence of J chain. The neutralization specific activity of the recombinant antibody produced in transiently or stably transformed cells was 50-fold higher than that of a monomeric rMAb with the IgG1 isotype and an identical binding site. This rMAb had titers of up to 10(4) by radioimmunoassay (RIA) and neutralized virus infectivity up to 10(4)-fold. Of 23 transgenic mice, 17 integrated both light and heavy chains, and at least 10 of them transmitted both genes to the progeny, leading to 100% of animals secreting functional TGEV neutralizing antibody during lactation. Selected mice produced milk with TGEV-specific antibody titers higher than 10(6) as determined by RIA, neutralized virus infectivity by 10(6)-fold, and produced up to 6 mg of antibody per ml. Antibody expression levels were transgene copy number independent and integration site dependent. Comicroinjection of the genomic beta-lactoglobulin gene with rMAb light- and heavy-chain genes led to the generation of transgenic mice carrying the three transgenes. The highest antibody titers were produced by transgenic mice that had integrated the antibody and beta-lactoglobulin genes, although the number of transgenic animals generated does not allow a definitive conclusion on the enhancing effect of beta-lactoglobulin cointegration. This approach may lead to the generation of transgenic animals providing lactogenic immunity to their progeny against enteric pathogens.

Amino Acid Sequence↗

Oral desensitisation in cow milk allergy: immunological findings.

In the literature there are several reports dealing with the possibility of a desensitising treatment in food allergy, but there are very few studies about the immunological mechanisms of oral desensitisation. We studied the immunological modifications in four children who underwent oral desensitisation with cow milk. Four children with cow milk allergy underwent oral desensitisation according to a standardized protocol. Total IgE, eosinophilic cationic protein in serum, and specific IgE and IgG4 to α-lactalbumin, to β-lactoglobulin and to casein were determined at the beginning of the treatment and after 6, 12 and 18 months in the 4 children treated. All the 4 treated patients successfully completed the treatment. Specific IgE to casein showed a significant reduction (p<0.01), while specific IgG4 to α-lactalbumin (p<0.02), to β-lactoglobulin (p<0.01) and to casein (p<0.01) showed a significant increase. Total IgE, eosinophilic cationic protein, and specific IgE to α-lactalbumin and to β-lactoglobulin did not show any significant modification. Control patients did not show any immunological modification and still had a positive double-blind, placebo-controlled food challenge. These results make us think that oral desensitisation in food allergy happens with the same mechanisms of traditional desensitising treatments for respiratory and insect sting allergies.

Journal Article↗

Linkage relationships among loci of polymorphisms in blood and milk of cattle.

Sequential probability tests were employed to examine genetic linkage among, cattle A, B, C, F, J, L, M, S, Z, and R' blood group loci, the hemoglobin locus, the serum transferrin, amylase, and alkaline phosphatase loci, and the milk protein loci beta-lactoglobulin, alpha S1-casein, beta-casein, and kappa-casein. Linkage was evident between the A and hemoglobin loci, the J and beta-lactoglobulin loci, the alpha S1-casein and beta-casein loci, the alpha S1-casein and kappa-casein loci, and the beta-casein and kappa-casein loci. Recombination fractions for these respective combinations were 0, .18, .03, .04, and .06. There was a suggestion of possible loose linkage (recombination fraction = .4) between the beta-lactoglobulin locus and the casein complex. Linkage could be excluded for most other combinations at .25 recombination.

Animals↗

Association of genetic variants of casein and milk serum proteins with milk, fat, and protein production by dairy cattle.

Polyacrylamide gel electrophoresis methods were used to phenotype caseins for 2045 cows and milk serum proteins for 3870 cows distributed in 63 Quebec dairy herds. Frequencies were: alpha s1-casein A .003, alpha s1-casein B .970, alpha s1-casein C .027; beta-casein A1 .561, beta-casein A2 .421, beta-casein A3 .011, beta-casein B .007; kappa-casein A .744, kappa-casein B .256; beta-lactoglobulin A .387, beta-lactoglobulin B .613; alpha-lactalbumin B 1.00. Overall unadjusted means for 305-day production in first lactation were: 5530 +/- 26.6 kg, 197 +/- 1.0 kg, 172 +/- 1.0 kg, 3.58 +/- .009, 3.12 +/- .009 for milk yield, fat yield, protein yield, fat percentage, and protein percentage for 1687 cows for casein systems. Similar data for 2906 cows phenotyped for milk serum proteins were: 5412 +/- 20.6 kg, 193 +/- .8 kg, 170 +/- .7 kg, 3.57 +/- .007, and 3.13 +/- 007. Least squares analysis of variance showed that herd location, month of calving, age of cow at first calving, and protein variants had significant effects on production traits. alpha s1-Casein B and beta-casein A phenotypes were associated with higher milk, fat, and protein yields than other variants in the two casein systems. Milk from kappa-casein BB and beta-lactoglobulin AA phenotypes contained .13 and .05% more protein than the AA and BB phenotypes with the AB phenotype intermediate.

Animals↗

Rapid separation of milk whey proteins by anion exchange chromatography.

A fast protein liquid chromatography system was used to fractionate the major proteins of sweet and acid wheys. Fifty to 500 microliter of whey were fractionated with a stepwise ionic strength gradient using water (buffer A) and increasing concentrations of .7 M sodium acetate (buffer B). Six well-resolved peaks were obtained: 1) amino acids (tentative identification), 2) low molecular weight peptides (tentative identification), 3) highly enriched alpha-lactalbumin, 4) highly enriched serum albumin 5) electrophoretically pure beta-lactoglobulin B, and electrophoretically pure beta-lactoglobulin A. A poor baseline or unresolved peaks resulted when .02 M bis-tris or .02 M histidine was used for buffer A and .7 M sodium acetate in .02 M bis-tris or histidine was used for buffer B. When sodium chloride was used in place of sodium acetate, beta-lactoglobulins A and B were poorly resolved. The column was cleaned after each run by injecting 2 ml of the following reagents: glacial acetic acid, 2 N sodium chloride, 2 N sodium hydroxide, 2 N sodium chloride, 2% detergent, and 100% acetonitrile. Time required to run each sample including column cleanup was 40 min.

Animals↗

Associations of bovine blood and milk polymorphisms with lactation traits: Guernseys.

First lactation milk yield and percentages of fat, solids-not-fat, and protein were analyzed in Guernsey cows to determine relationships of production traits to genetic markers at 16 polymorphic loci. The polymorphic systems examined were blood groups A, B, C, F, J, L, M, S, and Z; blood proteins transferrin, hemoglobin, and alkaline phosphatase; and milk proteins beta-lactoglobulin, alpha s1-casein, beta-casein, and kappa-casein. Different statistical models were utilized to evaluate direct genetic marker effects, linkage group effects, and heterozygosity effects. There were many indications of relationship with milk composition traits but for milk yield only for A system direct effects and for F system linkage effects. Pronounced associations between markers and component percentages were noted for the J, Z, and beta-lactoglobulin systems with fat percentage, for the M system with fat and solids-not-fat percentages, for the alkaline phosphatase system with solids-not-fat percentage and protein percentages, and for L and alpha s1-casein systems with protein percentage. Additionally, the interaction of beta-lactoglobulin and alpha s1-casein markers was significant for deviations of percent fat and percent solids-not-fat.

Animals↗

Enzymatic assay for the combined determination of plasmin plus plasminogen in milk: revisited.

The ability of beta-lactoglobulin variants A and B, alpha-lactalbumin, and BSA to inhibit plasmin plus plasminogen activity was examined. Data showed that beta-lactoglobulin A at concentrations of .2 and 1 mg/ml inhibited plasmin plus plasminogen activity by 18 and 54%. beta-Lactoglobulin B had no effect on plasmin plus plasminogen activity. At concentrations of .2 and 1 mg/ml, BSA inhibited plasmin plus plasminogen activity by 25 and 63%. alpha-Lactalbumin at concentrations of .2 and 1 mg/ml caused 1.9 and 20% inhibition of plasmin plus plasminogen activity. These data, collectively, suggest that existing methodology for measuring plasmin activity in milk serum underestimates real plasmin activity in milk. Underestimation is more pronounced in samples with high whey protein content (late lactation milk and milk obtained from mastitic quarters). To avoid this problem, we have modified the existing methodology. Our modification allows plasmin determination without interference from whey proteins and other plasmin inhibitors that are present in the serum fraction of bovine milk.

Aminocaproic Acid↗

[Biochemical and cytological changes in the milk and blood of cows with subclinical mastitis].

Some biochemical and cytological investigations were carried out with milk and milk serum, blood and blood serum of 87 cows, of which 57 manifested symptoms of subclinical mastitis and 25 were normal. It was established that the milk of animals having subclinical mastitis had lower content of lactose, inorganic phosphorus, and sialic acids; on the other hand chlorides were at a higher level, and the activity of alkaline phosphatase rose. Besides, the contents of serum albumin and gamma-lactoglobulins showed dependable rise, while those of alpha-lactoglobulin and beta-lactoglobulin dropped. Dependable was likewise the drop of the total protein level. However, no changes were found in the total protein content and the distribution of protein fractions in the blood serum of cows manifesting inflammation of the udder.

Animals↗

Polymorphism of molecular-genetic systems in the Polish red cattle.

The object of this study was to investigate gene polymorphism in the Polish Red (PR) cattle, a local Polish breed included in the FAO conservation programme. Milk protein genes and other genes with possible effect on production traits were analysed. Frequencies of different gene variants were compared with those in the Polish Friesian cattle. The following gene polymorphisms were analysed with PCR/RFLP technique: milk protein genes--kappa-casein and beta-lactoglobulin, growth hormone (GH), PitI (a transcription factor) and leptin. Moreover, SSCP analysis was performed of myostatin (MSTN) gene at the site previously shown to cause muscle overgrowth in Belgian Blue double-muscled cattle. A significant difference was found in this study between kappa-casein A and B allele frequency in PR and Friesian cattle. No such differences were found in the frequency of A and B alleles of beta-lactoglobulin, L and V alleles of GH, A and B alleles of PitI, and A, B and C alleles of leptin gene. In the analysed group of the Polish Red cattle three animals were found with the rare AI genotype of -lactoglobulin. No such genotype was identified in analysed Friesians. Moreover, 8 PR animals were identified carrying a mutation in MSTN gene, possibly identical to that causing the double-muscled phenotype in some breeds of meat cattle.

Animals↗

cDNA cloning and sequencing reveals human tear prealbumin to be a member of the lipophilic-ligand carrier protein superfamily.

The gene encoding human tear prealbumin, a major component of the protein fraction of tear fluid, was cloned from total cDNA of lacrimal gland by polymerase chain reaction using synthetic oligonucleotides derived from N-terminal amino acid sequences of the purified protein. Sequence analysis and a computer-assisted homology search revealed this protein to be a member of the lipocalin superfamily, consisting of hydrophobic-ligand carriers. The deduced amino acid sequence of tear prealbumin shares 58% identity with von Ebner's gland protein from rat, which is supposed to be involved in taste reception. In addition, significant homology has also been found with other members of the lipocalins, e.g. with beta-lactoglobulin. The predicted secondary structure of tear prealbumin resembles that of beta-lactoglobulin in the number and positions of nine beta-sheets and one alpha-helix at the C-terminal part of the protein, thus indicating a three-dimensional structure similar to beta-lactoglobulin. Protein sequencing revealed that the observed electrophoretic heterogeneity of tear prealbumin is due to subtle differences at the N terminus of the protein. The function of tear prealbumin as a lipophilic carrier was further supported by the fact that it binds [3H]retinol in vitro. Although this protein was originally described to be tear-specific, a tear prealbumin-specific antiserum also reacted with proteins of human saliva, sweat, and nasal mucus.

Amino Acid Sequence↗

[Disc electrophoretic characteristics of the serum proteins of "Vitalact"-type "humanized" milk].

Disc-electrophoreograms in the polyacrylamide gel of the cow milk serumal proteins show 11 peaks belonging to immune globulins, the poteose-peptone fraction, the serum-globulin, alpha-lactoalbumin, beta-lactoglobulin and to glucoproteids. Under a high-temperature treatment of humanized milk (105 degrees----10 minutes) a significant denaturation of the serumal proteins, re-distribution and a fall in the amount of proteinic fractions were noted. Beta-lactoglobulins and immune globulins are most sensitive. With disc-eletrophoresis in the polyacrylamide gel of the human milk serumal proteins the densitograms distinctly demonstrate the presence of peaks that correspond to immunoglobulins, proteose-peptones, serum-albumins, alpha-lacto-albumins in the absence of the beta-lactoglobulins fraction. The cited data allow the method of disc-electrophoresis in the polyacrylamide gel to be employed for improving the fractional composition of the serumal proteins in the nutrients intended for nurslings and infants of the first year of life.

Animals↗

Prenatal allergen contact with milk proteins.

BACKGROUND: Cellular proliferation to various allergens (Dermatophagoides pteronyssinus, beta-lactoglobulin, bovine serum albumin, ovalbumin) has been found in cord blood cells. Whether this reflects a sensitization during foetal life is uncertain. OBJECTIVE: We studied the cellular reactivity and cytokine production of cord blood cells in response to cow's milk proteins in a randomly selected group of newborns. The delineation of possible in utero allergen contact was attempted. METHODS: Cord blood mononuclear cells from 39 neonates were incubated with cow's milk proteins (alpha-lactalbumin, beta-lactoglobulin, casein, alpha-casein, beta-casein, kappa-casein, bovine serum albumin) for 7 days, and proliferation was assessed by incorporation of [3H]thymidine. Cord blood cell-derived interferon-gamma (IFN gamma) and interleukin-4 (IL-4) secretion was evaluated in response to allergen or phytohaemagglutinin (PHA) stimulation. RESULTS: A pronounced proliferation of cells stimulated with alpha-lactalbumin (ALA: mean stimulation index 8.0, 95% confidence interval 5.2-10.8), beta-lactoglobulin (BLG: mean stimulation index 5.9, 95% confidence interval 3.2-8.6) and alpha-casein (2.6, 95% confidence interval 2.9-9.1), as opposed to unstimulated cells in medium, was found. No correlation was found between cellular proliferation to milk proteins and parental atopy, maternal total IgE or cord blood IgE. IFN gamma production (but not IL-4) was inducible by PHA (range 429-1810 pg/ml), but only in one individual upon stimulation with BLG. Preferentially, reduced IFN gamma levels were found in individuals with positive parental allergic history. CONCLUSION: The recognition of allergen by cord blood cells indicates that allergen priming must occur prenatally. The relevance for subsequent sensitization is unclear.

Allergens↗

Rapid separation and quantification of major caseins and whey proteins of bovine milk by capillary electrophoresis.

A rapid capillary zone electrophoresis (CZE) method was established for separating and quantifying major casein and whey proteins in milk. Optimum sample preparation and electrophoretic conditions in a coated capillary maintained at 40 degrees C allowed accurate and reproducible quantification of milk proteins in a single analysis. Sample and run buffer allowed caseins to be maintained in solution by using a combination of urea and a nonionic detergent in phosphate buffer at pH 2.5. Quantitative CZE protein data were derived by calculating percentages and concentrations (mg/mL) of alpha-casein, beta-casein, alpha-lactalbumin, and beta-lactoglobulin. Calibration curves followed linear relationships with highly significant (p < 0.1) correlation coefficients. Relative standard deviations of less than 0.82 (%) for migration times and 2.18 (%) for percent protein indicated that the technique was reproducible. Electrophoretic protein profiles of fresh bovine milk and rehydrated dry milk showed marked quantitative differences in whey protein concentrations. Whey protein represented 12.37 +/- 0.07% beta-lactoglobulin and 3.05 +/- 0.08% alpha-lactalbumin of total protein in typical fresh milk, while only 1.90 +/- 0.16% beta-lactoglobulin and 0.86 +/- 0.04% alpha-lactalbumin of total protein were detected in a commercial rehydrated milk powder. By quantifying these differences, the established technique may allow the detection of substitution of fresh milk with rehydrated milk powder. The accuracy and reproducibility of the technique permitted the quantitation of individual protein concentrations in milk samples, which agreed with ranges reported in the literature. CZE may be well suited for routine use by dairies and regulatory agencies, since it allows the determination of milk proteins in less than 60 min.

Animals↗

PROTEINS OF RUMINANT COLOSTRUM.

1. The proteins in the whey of ewe colostrum resemble those obtained from goat colostrum in containing two main electrophoretic components, with mobilities of -2x10(-5) and -3.9x10(-5) cm.(2)sec.(-1)v(-1) in sodium veronal buffer, pH 8.6 and I 0.1, at 1 degrees . 2. Of 32 samples of goat colostrum examined by paper electrophoresis 27 contained the immune lactoglobulin characteristic of colostrum; of 55 samples of cow colostrum all contained immune lactoglobulin.

Animals↗

PROTEINURIA OF NEWBORN SUCKING RUMINANTS.

1. Proteinuria was found in most sucking lambs and calves examined but was less prevalent in bottle-fed kids or bucket-fed calves. 2. This incidence of proteinuria corresponded to the presence of immune lactoglobulin in the serum. 3. The extent of proteinuria in lambs and calves rose to 20 g./l. or more, whereas in kids values of only 4 g./l. were obtained. 4. The proteins present in the urine of lambs and kids have been compared with those in the urine of calves by using boundary electrophoresis and paper electrophoresis. 5. Sedimentation analyses have been made on the proteins of lamb, kid and calf urine. Components with sedimentation coefficients 3s and less were present. The separated slow electrophoretic components were found to have sedimentation coefficients 3.3, 3.6 and 3.3s respectively. 6. The sedimentation analyses, and for calf urine the electrophoretic data, support the view that the slow-moving electrophoretic components of calf and lamb urine are degraded immune lactoglobulins. 7. There was electrophoretic evidence for other degraded proteins in some lamb urine which showed multiple zones that could not be related to serum or colostral proteins.

Animals↗

Investigation of some covalent and noncovalent complexes by matrix-assisted laser desorption/ionization time-of-flight and electrospray mass spectrometry.

Covalent and noncovalent complexes involving bovine superoxide dismutase, bovine alpha-lactalbumin and two variants, A and B, of bovine beta-lactoglobulin have been examined by delayed extraction matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and electrospray mass spectrometry. Covalent complexation with acrylamide was obtained through the interaction of these proteins with acrylamide monomers while treatment with peroxynitrite yielded complexes with NO2. Some of the complexation sites with acrylamide were reliably identified by performing tryptic digestion followed by MALDI-TOF measurements in the reflection mode. These data demonstrate that the presence of free cysteine in the investigated sequences is not a precondition for the observation of Cys-acrylamide complexes. Although the bulk of the present work is dedicated to the characterization of Cys-acrylamide adducts, the preliminary data on noncovalent complexes between two of those proteins and 8-anilinonaphthalene-1-sulfonic acid (ANS) were easily observed under electrospray conditions, while under MALDI-TOF conditions only the complex with beta-lactoglobulin B was clearly evident. The presented data demonstrate the advantage of the parallel use of both ionization techniques for this type of investigation.

Acrylamide↗

Alginate as a displacer for protein displacement chromatography.

Alginate use in displacement chromatography as a displacer has been studied. The experiments showed that untreated alginate is the basis of potential displacer for displacement chromatography, but needs to be cleaved into smaller chains. Alginate treated with ultrasound, which cleaves alginate into shorter polysaccharide chains, gave better displacement than untreated alginate, while alginate subjected to limited acid hydrolysis gave the best results in displacement chromatography. It was found that the mixture of ovalbumin and beta-lactoglobulin separated well, and several components of ovalbumin were also separated and purified when alginate hydrolysate was used as a displacer. beta-Lactoglobulins A and B, which have the same molecular weight and differ in isoelectric point by only 0.1 pH units, were displaced from Q-Sepharose by alginate hydrolysate.

Alginates↗