Determination of β-lactoglobulin in Anti-HPV Biological Protein Dressing by Amino Acid Assay / 中国医疗器械杂志

OBJECTIVE@#To establish an amino acid assay for the determination of β-lactoglobulin in Anti-HPV biological protein dressing.@*METHODS@#Under acidic conditions, β-lactoglobulin is hydrolyzed into free amino acids, separated by cation exchange chromatography, and derivatived after ninhydrin column. The chromatogram at 570 nm is collected. The content of β-lactoglobulin in the sample is indirectly determined by measuring the lysine content obtained by hydrolysis.@*RESULTS@#β-lactoglobulin has a good linear relationship in the concentration range of 77.28~309.12 μg/mL (@*CONCLUSIONS@#The method is simple, specific, accurate and reproducible, which is suitable for the quantitative analysis of β-lactoglobulin in anti-HPV biological protein dressing.

Self-assembly in the transparent droplets formed during the screening of protein self-assembly conditions / 生物工程学报

Protein self-assemblies at the micro- and nano-scale are of great interest because of their morphological diversity and good biocompatibility. High-throughput screening of protein self-assembly at different scales and morphologies using protein crystallization screening conditions is an emerging method. When using this method to screen protein self-assembly conditions, some apparently transparent droplets are often observed, in which it is not clear whether self-assembly occurs. We explored the interaction between β-lactoglobulin and the protein crystallization kit Index™ C10 and observed the presence of micro- and nano-scale protein self-assemblies in the transparent droplets. The diverse morphology of the micro- and nano-scale self-assemblies in the transparent droplets formed by mixing different initial concentrations of β-lactoglobulin and Index™ C10 was further investigated by scanning electron microscope. Self-assembly process of fluorescence-labelled β-lactoglobulin was monitored continuously by laser confocal microscope, allowing real-time observation of the liquid-liquid phase separation phenomenon and the morphology of the final self-assemblies. The internal structure of the self-assemblies was gradually ordered over time by in-situ X-ray diffraction. This indicates that the self-assembly phenomenon within transparent droplets, observed in protein self-assembly condition screening experiments, is worthy of further in-depth exploration.

Polymorphisms of leptin, beta-lactoglobulin and pituitary transcription factor have no effect on milk characteristics in crossbred cows / Polimorfismos da leptina, beta-lactoglobulina e fator de transcrição da hipófise não afetam as características do leite em vacas mestiças

O objetivo deste trabalho foi identificar polimorfismos genéticos de leptina, ß-lactoglobulina e fator de transcrição pituitária (PIT1) e avaliar seus efeitos na composição química e na contagem de células somáticas de leite de vacas leiteiras mestiças que vivem em um clima quente. Um total de 291 vacas leiteiras mestiças foram investigadas. Foram coletadas amostras de sangue para extração de DNA e amostras de leite. As amostras foram classificadas em três grupos genéticos: 12/ (42), 34/ (83) e 78/ (166) Holandês x Guzerá. As frequências de alelos e genótipos foram determinadas e o equilíbrio Hardy-Weinberg foi avaliado. Foram realizadas análises da composição do leite (gordura, proteína, lactose e extracto seco desengordurado), contagem de células somáticas e rendimento leiteiro. Os grupos genéticos e os polimorfismos genéticos para cada gene foram utilizados como efeitos fixos na análise. O único polimorfismo encontrado em equilíbrio de Hardy-Weinberg foi para o genótipo da ß-lactoglobulina. No presente estudo, era esperado que a maioria das variáveis de composição variasse entre os genótipos. Já se sabe que os cruzamentos dão origem a animais com características fenotípicas e genotípicas. No entanto, os polimorfismos não influenciaram a composição e a qualidade do leite nas vacas 12/ , 34/ e 78/ Holstein x Guzerá mantidas em um clima quente.(AU)

Factor analysis as a tool to estimate association among individual proteins and other milk components with casein micelle size and cheese yield / Análise fatorial como ferramenta para estimar a associação entre proteínas individuais e outros componentes do leite com o tamanho das micelas de caseína e massa seca de queijo

The present study attempted to identify individual milk proteins and other milk components that are associated with casein micelle size (CMS) and dry matter cheese yield (DMCY) using factor analysis. Here, we used 140 bulk tank milk samples from different farms. Milk composition was determined using a Fourier transform infrared equipament. The individual milk proteins were (αS-casein, ß-casein, κ-casein, ß-lactoglobulin and α-lactoalbumin) measured by their electrophoretic profile. The CMS was estimated by photon correlation spectroscopy, and the DMCY was determined using reduced laboratory-scale cheese production. Factor analysis partitioned the milk components into three groups that, taken together, explain 68.3% of the total variance. The first factor was defined as "CMS", while the second as "DMCY" factor, based on their high loadings. The CMS was positively correlated with protein, casein, non-fat solids and αS-casein and negatively associated with κ-casein and ß-lactoglubulin. DMCY was positively correlated with fat, protein, casein, total solids and negatively correlated with αs-casein. These results indicate that the variation of individual milk proteins may be an important aspect correlated to milk quality and cheese production.(AU)

O objetivo do presente estudo foi avaliar a associação das frações proteicas individuais e de outros componentes do leite com o tamanho das micelas de caseína (TMC) e a produção de matéria seca de queijo (MSQ) utilizando-se análise fatorial. Foram coletadas 140 amostras de leite de tanque provenientes de diferentes fazendas. A determinação da composição do leite foi determinada por espectroscopia no infravermelho com transformação de Fourier. As proteínas individuais (αS-caseína, ß-caseína, κ-caseína, ß-lactoglobulina e α-lactalbumina) foram quantificadas pelo perfil eletroforético. O tamanho médio das micelas de caseína foi analisado pelo princípio de espectroscopia de correlação de fótons e pela produção MSQ a partir do modelo de coagulação do leite em escala reduzida. A análise fatorial delimitou as variáveis em três fatores, que, juntos, responderam por 68,3% da variação total dos dados. No primeiro fator foram observadas as associações mais fortes com o TMC, enquanto no segundo fator as correlações foram mais significativas com a MSQ. O TMC foi associado positivamente com o conteúdo de proteína, caseína, sólidos desengordurados e αS-caseína, e negativamente com κ-caseína e ß-lactoglubulina. MSQ foi associada positivamente com o teor gordura, proteína e caseína total, sólidos totais, e negativamente com o teor de αs-caseína. Esses resultados indicam que a variação quantitativa das proteínas do leite pode ser determinante da qualidade do leite na produção de queijo.(AU)

In vitro culture of ovine mammary gland cells expressing beta-lactoglobulin and beta-casein / Células da glândula mamária ovina cultivadas in vitro expressam beta-lactoglobulina e beta-caseina

The expression of milk proteins in vitro is essential to exploit the mammary gland cells as a biological model. Enzymatic tissue disaggregation has been widely used to establish mammary cell culture, but its effect in long-term ovine mammary cell culture is not completely elucidated. This study aimed at comparing mechanical/enzymatic and mechanical dissociation methods to establish ovine mammary cell culture. We compared cellular differentiation induced by lactating ewe serum or fetal bovine serum based on the gene expression levels of milk proteins (beta-lactoglobulin, alpha s1-casein, and betacasein). Mechanically dissociated cells were positive immunostaining for cytokeratin 8.13, such as mammary epithelial cells. These cells are responsible for milk protein expression and they are low immunostaining for vimentin, mesenchymal marker. Mechanical/enzymatic dissociation cells were positive for vimentin. The fastest cell growth (cell/hour) was observed in the mechanical dissociation group cultured with 10% fetal bovine serum medium. Mechanically and mechanically/enzymatically derived cells were able to express beta-casein and beta-lactoglobulin, but not alpha s1-casein. The relative expression of beta-lactoglobulin was not affected by the tissue dissociation method or culture media, beta-casein relative expression was down regulated in mechanically dissociated cells cultured in the presence of lactating ewe serum, (P = 0.019). Beta-casein relative expression was also down regulated in mechanically/enzymatically dissociated cells cultured with fetal bovine serum (P = 0.021). In the present conditions, we conclude that mechanical dissociation followed by culture with 10% of fetal bovine serum was the most efficient method to induce milk proteins' mRNA expression by ovine mammary epithelial cells in vitro.(AU)

A expressão in vitro de proteínas do leite é essencial para explorar as células da glândula mamária como um modelo biológico. A desagregação tecidual via enzimática é amplamente utilizada para o estabelecimento cultivo de células mamárias. No entanto, seu efeito a longo prazo no cultivo de células da glândula mamária ovina ainda não é bem elucidado. Este estudo tem como objetivo comparar dois métodos de dissociação tecidual, mecânico/enzimático e mecânico, para estabelecer cultivo celular de glândula mamária ovina. A indução da diferenciação celular, por adição de soro de ovelha lactante ou soro fetal bovino, foi avaliada pelos níveis de expressão de proteínas do leite (beta-lactoglobulina, alpha s1-caseína e beta-caseína). Células mecanicamente dissociadas foram positivamente marcadas para a presença de citoqueratina 8.13, marcador para células epiteliais mamárias. Essas células são as responsáveis pela produção das proteínas do leite e são pouco marcadas para a presença de vimentina, marcador para células de origem mesenquimal. Já as células obtidas da dissociação mecânica/ enzimática foram positivamente marcadas para presença de vimentina. A maior velocidade de crescimento (células/hora) foi observado para o grupo com dissociação mecânica cultivado em meio com 10% de soro fetal bovino. As células obtidas tanto da dissociação mecânica quanto mecânica/enzimática foram capazes de expressar beta-lactoglobulina e beta-caseína, mas não alfa s1-caseína. A expressão relativa de beta-lactoglobulina não foi afetada pelo método de dissociação ou meio de cultivo. A expressão relativa da beta-caseína foi negativamente regulada para células mecanicamente dissociadas e cultivadas na presença de soro de ovelha lactante (P = 0,019). A expressão relativa da beta-caseína também foi negativamente regulada para células dissociadas de forma mecânica/enzimática e cultivadas com soro fetal bovino (P = 0,021). Nas condições do presente estudo, concluímos que o método de dissociação mecânica seguido pelo cultivo em meio com 10% de soro fetal bovino foi o método mais eficiente para induzir a expressão mRNA de proteínas do leite por células epiteliais mamárias ovinas in vitro.(AU)

BLG gene knockout and hLF gene knock-in at BLG locus in goat by TALENs / 生物工程学报

To knock out β-lactoglobulin (BLG) gene and insert human lactoferrin (hLF) coding sequence at BLG locus of goat, the transcription activator-like effector nucleases (TALEN) mediated recombination was used to edit the BLG gene of goat fetal fibroblast, then as donor cells for somatic cell nuclear transfer. We designed a pair of specific plasmid TALEN-3-L/R for goat BLG exon III recognition sites, and BLC14-TK vector containing a negative selection gene HSV-TK, was used for the knock in of hLF gene. TALENs plasmids were transfected into the goat fetal fibroblast cells, and the cells were screened three days by 2 μg/mL puromycin. DNA cleavage activities of cells were verified by PCR amplification and DNA production sequencing. Then, targeting vector BLC14-TK and plasmids TALEN-3-L/R were co-transfected into goat fetal fibroblasts, both 700 μg/mL G418 and 2 μg/mL GCV were simultaneously used to screen G418-resistant cells. Detections of integration and recombination were implemented to obtain cells with hLF gene site-specific integration. We chose targeting cells as donor cells for somatic cell nuclear transfer. The mutagenicity of TALEN-3-L/R was between 25% and 30%. A total of 335 reconstructed embryos with 6 BLG-/hLF+ targeting cell lines were transferred into 16 recipient goats. There were 9 pregnancies confirmed by ultrasound on day 30 to 35 (pregnancy rate of 39.1%), and one of 50-day-old fetus with BLG-/hLF+ was achieved. These results provide the basis for hLF gene knock-in at BLG locus of goat and cultivating transgenic goat of low allergens and rich hLF in the milk.

Polymorphism of three milk protein genes in Mexican Jersey cattle

The objective was to estimate the allelic and genotypic frequencies, genetic diversity and polymorphic information content for the β-casein, κ-casein and β-lactoglobulin genes. Blood and frozen semen samples were collected from 453 Jersey individuals registered by the Mexican Jersey Cattle Association. Twenty eight breed specific SNP primers for whole genes were used. The B allele of κ-casein had higher frequency (0.69) than the A (0.26) and E (0.05). For β-lactoglobulin, the highest frequency was for B (0.72), followed by A and C alleles (0.26 and 0.02, respectively). The β-casein allele with the highest frequency was A² (0.71), followed by A¹ (0.19), A³ (0.05), B (0.04) and C (0.01). The average genetic diversity (He) was 0.53. The average locus effective allele number was 1.79. These results indicate a high allelic diversity for κ-caseín, β-casein and β-lactoglobulin that could be included in breeding programs in the population studied, aimed to improve the milk quality traits of economic importance.

Molecular study of some candidate genes affecting milk production traits in Holstein cattle

Most of the economically important traits in dairy cattle are quantitative in nature, which means that they are affected by environmental factors and by large number of gene. Selection of superior animals has been made more effective through studies of major milk protein genes that are known to affect both milk yield and composition. The present study was carried out to detect polymorphism in kappa casein [CSN3] and beta-lactoglobulin [LGB] genes in Holstein cattle under Egyptian condition through DNA sequencing and polymerase chain reaction restriction fragment length polymorphism [PCR-RFLP]. Fifty Animals were divided into high and low milk producing according to their breeding value. PCR amplification of exon III o CSN3 and LGB was performed followed by restriction fragment length polymorphism [PCR-RFLP] using Hind-III and HinfI for CSN3 and HaeIII restriction endonuclease for LGB. Nucleotide polymorphisms between high and low producing cows were detected by DNA sequencing. The restriction enzymes digestion failed to produce restriction patterns and revealed no polymorphism in all studied animals. Comparison of nucleotide sequences between high and low producing cows revealed lack of polymorphism in CSN3 and four nucleotide changes in LGB gene; C179 T, C225T, T246C, and C294G. The further study using other specific restriction endonuclease was required to detect polymorphisms of CSN3 and LGB in Egyptian Holstein cattle. SNPs discovered in this study can be used as molecular genetic markers for marker assisted selection [MAS] to increase and accelerate the rate of genetic improvement of milk production traits

The effect of heat treatment or hydrolysis on cow's milk protein distributions and antigenicities

PURPOSE: Cow's milk protein is one of the most common and strongest food allergen. We investigated the effects of heat treatment on the distribution and antigenicities of major allergens from cow's milk. We also compared the protein distribution and antigenicities among cow's milk formula and its substitutes. METHODS: We heated alpha-casen, beta-lactoglobulin (BLG), alpha-lactalbumin (ALA), and crude extract of cow's milk in 100degrees C boiling water for 1 hour. We prepared crude extracts from cow's milk formula, partially hydrolyzed milk formula (pHF) and extensively hydrolyzed milk formula (eHF). The protein compositions of all the samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The antigenicities were determined by IgE immunoblotting with pooled serum collected from 11 patients with milk allergy. RESULTS: After heating, no significant alteration was found in casein, and the aggregates of ALA and BLG were detected with molecular weights of about 30 and 45 kDa, respectively. The antigenicities of newly detected aggregates were increased. The new aggregates of BLG with increased antigenicities were also found in heated milk total protein. Major milk allergens were not found in pHF, and residual components with a molecular weight below 10 KDa did not show IgE-binding activity. We failed to observe the residual components and antigenicities of eHF. CONCLUSION: Changes in protein distribution and antigenicity of milk total protein induced by heat treatment may not be significantly different from those of each major allergen. The residual components of pHF could have little IgE-binding capacity, and there may be few or no antigenic components in eHF.

Polimorfismos genéticos da kappa-caseína e da beta-lactoglobulina e produção de leite em bovinos / Kappa-casein and beta-lactoglobulin genetic polymorphisms and milk yield in bovines

As variantes gênicas da beta-lactoglobulina (β-LG) e da kappa-caseína (κ-CN) bovinas são associadas à produção, qualidade e características de processamento do leite. O objetivo deste trabalho foi analisar as frequências dos genótipos AA, AB e BB, por meio da técnica de PCR-RFLP, da β-LG e da κ-CN bovinas, e suas associações com a produção de leite (kg leite/dia) em bovinos das raças Girolanda, Holandesa e Jersey. Para a κ-CN, a frequência do genótipo AA foi maior nos animais das raças Holandesa (37%) e Girolanda (63%). Na raça Jersey, houve predomínio do genótipo BB (60%). Para a β-LG, o genótipo AB foi o mais encontrado nas raças Girolanda (54%) e Holandesa (58%), enquanto nos animais da raça Jersey houve predomínio do genótipo BB (45%). Houve associação do alelo B da κ-CN com maior produtividade leiteira nas raças Girolanda e Holandesa, e do alelo A da β-LG com maior produtividade de leite na raça Jersey. As variantes genéticas da κ-CN podem ser usadas como marcadores na seleção para a produtividade leiteira nas raças Girolanda e Holandesa. Para a raça Jersey, as variantes da β-LG seriam mais adequadas para essa seleção.

Bovine beta-lactoglobulin (β-LG) and kappa-casein (κ-CN) genic variants are associated with productivity, quality and processing features of milk. The objective of this study was to analyze through the PCR-RFLP technique, the frequency of AA, AB and BB genotypes of bovine β-LG and κ-CN, and their association to milk production (kg milk/day) in Girolanda, Holstein and Jersey cattle. For κ-CN, the frequency of the AA genotype was higher in Holstein (37%) and Girolanda (63%), while there was a predominance of the BB genotype in Jersey (60%). For β-LG, the BB genotype was the most found in Girolanda (54%) and Holstein (58%), while there was a predominance of the BB genotype (45%) in Jersey. There was a positive association between B allele of κ-CN and milk production in the Girolanda and Holstein cattle and between A allele of β-LG and milk production in the Jersey cattle. Genetic variants of κ-CN could be used as markers for the selection for productivity in Girolanda and Holstein cattle. The genetic variants of β-LG would be more appropriate for this selection in the Jersey breed.

In search of a tolerance-induction strategy for cow's milk allergies: significant reduction of beta-lactoglobulin allergenicity via transglutaminase/cysteine polymerization

OBJECTIVE: To explore the use of β-lactoglobulin polymerized using microbial transglutaminase and heating to identify whether protein polymerization could reduce in vivo allergenicity and maintain in vitro and ex vivo immunoreactivity for use in tolerance-induction protocols. METHODS: Based on previous protocols applied in mice and children, we performed in vivo challenges (using a skin prick test) with native and polymerized β-lactoglobulin in adult patients with an IgE-mediated allergy to plactoglobulin. In vitro humoral immunoreactivity was analyzed using immunoblotting. Cell-mediated immunoreactivity was analyzed using ex vivo challenges with native and polymerized β-lactoglobulin and monitored by leukocyte adherence inhibition tests. RESULTS: The skin tests demonstrated that there was a significant reduction in immediate cutaneous reactivity after polymerization. Polymerization did not decrease the immunoblotting detection of s-IgE specific to β-lactoglobulin. Cell-mediated immunoreactivity, as assessed by ex vivo challenges and leukocyte adherence inhibition tests, did not exhibit significant differences between leukocytes challenged with native versus polymerized β-lactoglobulin. CONCLUSIONS: The polymerization of β-lactoglobulin decreased in vivo allergenicity and did not decrease in vitro humoral or ex vivo cell-mediated immunoreactivity. Therefore, we conclude that inducing polymerization using transglutaminase represents a promising technique to produce suitable molecules for the purpose of designing oral/ sublingual tolerance induction protocols for the treatment of allergies.

In vitro refolding process of bovine allergen β-lactoglobulin by Multispectroscopic method / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To characterize the relationship between the refolding process of recombinant bovine β-lactoglobulin and its immunoreactivity for clinical purposes. To establish a spectral method which examine the extent of recombinant allergen renaturation.</p><p><b>METHODS</b>The refolding process of recombinant bovine β-lactoglobulin was investigated by using circular dichroism, fluorescence and synchronous fluorescence spectra. IgE-binding capacity of recombinant protein was analyzed by ELISA. In addition, bioinformatic methods were used to explain the spectral characteristics and analyze the relationship between the conformational changes and the immunoreactivity of the protein during renaturation in vitro.</p><p><b>RESULTS</b>Renaturation of recombinant bovine β-lactoglobulin resulted in a more compact structure resembling the natural counterpart with stronger IgE-binding capacity.</p><p><b>CONCLUSION</b>The degree of protein renaturation correlated with the IgE-binding capacity of the protein. Results from this study may be of help for food allergy therapy and development of vaccination in the future.</p>

The effect of genetic polymorphism of the exon 2 of the beta-lactoglobulin gene on the milk composition in Chinese Holstein / 中国应用生理学杂志

<p><b>OBJECTIVE</b>In order to study the effect of the polymorphism at the exon2 region of the (3-LG allele gene on milk composition and yield.</p><p><b>METHODS</b>The single-strand conformation polymorphism method (PCR-SSCP) was used to analyze for polymorphism the exon2 region of the 3-LG gene (NCBI accession number: DQ489319) in Chinese Holstein.</p><p><b>RESULTS</b>Eight SSCP patterns were detected in the fragments: ab, abc, abd, abe, abcd, abce, abde and abcde, and the patterns frequencies as follows: 0.14, 0.10, 0.27, 0.23, 0.05, 0.04, 0.11 and 0.06 (P < 0.05); Six single nucleotide polymorphism (SNP) were detected in this study: sitel C>T, site2 T>C, site3 C>T, site4 C>C, site5 C> A, site6 A>T or C, and the polymorphism infonnation content (PIC) of these SNPs were in median or high polymorphism (PIC > 0.25).</p><p><b>CONCLUSION</b>These SNPs at the exon2 region of the beta-LG gene were remarkably and affected milk performance traits (milk yield, protein and fat contents) in Chinese Holstein.</p>

Reproducción y fertilidad humana: aspectos biomédicos de la familia de las lipocalinas: biología, patobiología y bioclínica / Human reproduction and fertility: biomedical aspects of the lipocalin family: biology, pathobiology and bioclinic aspects

La familia de genes de las lipocalinas (LCN) está compuesta por varios miembros que comparten una estructura común y que se han duplicado en forma repetida durante la evolución expandiéndose a más de 150 genes conocidos, de ellos al menos veinte reportados en la especie humana. El grupo de proteínas de las LCN está constituido por varios elementos que comparten la propiedad común de unión de ligandos lipofílicos. Las LCN funcionan en un amplio rango de sistemas incluyendo quimiorrecepción y transporte en fisiología sensorial del gusto y olor, coloración, modulación hemato-inmune, síntesis de prostanglandina D2, neuro-fisiología, fisiología reproductiva y fertilidad, embriogénesis, proliferación y división celular, supervivencia y apoptosis celular. Es evidente su rol en patobiología y bioclínica reproductiva y de la fertilidad al observar que varias LCN tienen niveles alterados de expresión en diferentes eventos patofisiológicos. Esta revisión resume hallazgos e implicaciones

The family of the lipocalin (LCN) genes is composed by various members that share a common structure and haveduplicated repeatedly during evolution expanding to more than 150 known genes of which at least 20 have been reported in the human species. The group of lipocalin-related proteins is comprised by a number of elements which share various common properties such as binding to different lipophilic ligands. Lipocalins are involved in a broad range of systems including chemoreception and transport functions in sensory physiology of taste and smell, coloration, modulation of hemato-immune response, synthesis of prostaglandin D2, neurophysiology, physiology of reproduction and fertility, embryogenesis, cell proliferation and division, and cell survival and apoptosis. Its role is evident in the pathophysiology and bioclinical aspects of reproduction and fertility consistent with the observation of altered levels of expression of several lipocalins in different pathophysiological events. This review summarizes findings and implications on this topic.

[Allergic reaction of goats and cows milk proteins]

Systemic anaphylaxis test in mouse showed that the visual effect of injection of cow casein, whey, casein fraction and bovine beta-lactoglobulin was strong. Similar results were found when goat casein, Kappa-casein and beta-casein were injected. The visual effect of injection of beta-lactoglobulin and alpha[s],-casein was low. No response was found when bovine and goat alpha-Lactalbumin was injected in animals. Passive cutaneous anaphylaxis test in mouse showed that the reaction area of cow casein injection was 0.63 cm[2] in comparison with 0.19 cm[2] for goat casein. The reaction of cow beta-lactoglobulin injection was 0.12 cm[2], while no reaction was occurred when goat beta-lactoglobulin, goat and cow alpha-lactalbumin were injected. The percentage of degranulation of mast cells when treated with cow raw milk, casein, whey, beta-lactoglobulin and alpha-lactalbumin were 32.11, 100, 41.80, 90.01 and 12.73% respectively, In comparison with 14.33, 80.19, 34.73, 39.57 and 10.86% respectively for the same proteins in goat milk

[Study of the immunological cross-reactions between goats and cows milk proteins]

Immunoelectrophoresis analysis showed immunological cross reactions between goat and cow milk caseins which belong to beta-casein, however, no such reaction were observed between goat and cow beta-lactoglobulin and alpha-lactalbumin. Systemic anaphylaxis test in guinea pigs showed strong immunological reactions between goat and cow milk proteins, injection of cow milk in animal's vein, which fed on cow milk caused 100% mortality. Same results were also obtained with injection of goat milk. Passive hemagglutination test against goat and cow milk was used to estimate antibody titer in guinea pigs serum, which fed cow's milk. The obtained results showed that the highest titer was found against casein followed by beta-lactoglobulin and alpha-lactalbumin for cow milk proteins, while for goat milk proteins the highest titer was found against casein followed by alpha-lactalbumm and beta-lactoglobulin. The titer of antibodies against goat alpha[s]-casein and Kappa-casein was lower than that for cow milk, the behavior of goat and cow beta-casein was similar for both proteins

Analysis of cow's milk specific IgE positive patients in newborns / 소아과

PURPOSE: The purpose of this study was to research whether measurement of cow's milk specific IgE on the newborn would be helpful in the diagnosis of cow's milk allergy. We tried to find out the relation between cow's milk specific IgE and other allergy diseases by following up cases. METHODS: We reviewed clinical features of 87 episodes in infants less than 4 weeks old who were positive in cow's milk specific IgE test. For the study group, history taking, physical examinations, elimination and cow's milk specific IgE tests were carried out. We investigated the connection among cow' milk specific IgE, allergic disease and family history in 40 of 87 patients we could follow up on. RESULTS: The mean age of the study group was 17.2+/-5.4 days. The subjects were classified in four groups according into allergens : 87 milk allergy positive patients, 24 casein positive, 38 alpha-lactoalbumin positive, and 75 beta-lactoglobulin positive. The number of patients who had follow-ups for more than 6 months to was 40(45.9 percent). The patients whose parents had allergic disease numberred 10(25 percent). Fiften patients had allergic diseases, 4 had asthma and 11 atopic dermatitis. According to the follow-up study, there is a significant relation between casein positive patients and allergic disease. But there is no statistical and significant relation between cow's milk specific IgE and a family history of allergic disease. CONCLUSION: For the newborn babies, elimination tests and cow's milk specific IgE tests can be useful in the diagnosis of IgE-mediated or mixed milk allergies.

Improvement of bovine ß-lactoglobulin production and secretion by Lactococcus lactis

The stabilizing effects of staphylococcal nuclease (Nuc) and of a synthetic propeptide (LEISSTCDA, hereafter called LEISS) on the production of a model food allergen, bovine ß-lactoglobulin (BLG), in Lactococcus lactis were investigated. The fusion of Nuc to BLG (Nuc-BLG) results in higher production and secretion of the hybrid protein. When LEISS was fused to BLG, the production of the resulting protein LEISS-BLG was only slightly improved compared to the one obtained with Nuc-BLG. However, the secretion of LEISS-BLG was dramatically enhanced (~10- and 4-fold higher than BLG and Nuc-BLG, respectively). Finally, the fusion of LEISS to Nuc-BLG resulting in the protein LEISS-Nuc-BLG led to the highest production of the hybrid protein, estimated at ~8 æg/ml (~2-fold higher than Nuc-BLG). In conclusion, the fusions described here led to the improvement of the production and secretion of BLG. These tools will be used to modulate the immune response against BLG via delivery of recombinant lactococci at the mucosal level, in a mouse model of cow's milk allergy.

Development of transgenic mice harboring ovine beta lactoglobulin-calcitonin transgene

Expression of foreign proteins in mammalian milk is becoming a widespread strategy for high-level production of recombinant pharmaceuticals, especially those with the most complex post-translational modifications. We have attempted in this project to develop transgenic mice harboring a transgene driving mammary gland expression of hybrid human salmon calcitonin. A milk-specific ovine beta-lactoglobulin [oBLG] promoter was used to drive expression of recombinant calcitonin in mouse milk. A gene construct was generated, consisting of 10.7kbp of the oBLG gene including its promoter and 3 flanking region with the calcitonin coding sequences inserted in-frame into the oBLG fifth exon. The gene construct was purified using CsCl gradient, released from vector, and gel-purified. After appropriate dilution, it was microinjected into recently-fertilized mouse oocytes. These oocytes then were transferred to pseudo-pregnant foster mice. Forty one pups were born from foster mice, which were genotyped using PCR, slot blotting, and Southern blotting. Among 9 mice which showed positive PCR results, 6 mice resulted in pups with positive PCR tests. All six families transmitted the transgene to first and second generation. As the main criteria for considering a mouse as transgenic is transgene transmission to the next generation, all 6 mice which stably transmitted their transgene to progeny are considered as transgenic founders and constitute independent transgenic lines

Cow's Milk Protein-specific IgE Concentrations in Two Age Groups of Children with cow's Milk Allergy / 소아알레르기및호흡기학회지

BACKGROUND: About 70-80% of children with cow's milk allergy (CMA) become outgrown clinically by the age of 3 years. Casein, one of the three major cow's milk proteins (casein, beta-lactoglobulin (BLG), alpha-lactoalbumin (ALA) ) has been reported to play an important role in the persistence of CMA. The aim of this study was to determine different effects of causative milk proteins on the persistence of CMA between two age groups. METHODS: A total of 65 patients with CMA were enrolled in this study. Their cow's milk-specific IgEs were positive ( 0.7 U/ml by Pharmacia CAP). After dividing 65 patients into two age groups, under the age of 3 years and over 3 years (persistent CMA), we compared the levels of casein-, BLG- and ALA-specific IgE antibodies between the two groups. RESULTS: There were 44 patients in the group of less than 3 years of age and 21 patients in the group of more than 3 years of age. The concentrations of the specific IgE antibodies to casein, BLG and ALA were not significantly different between the two groups. However, although statistically insignificant, those more than 3 years of age had higher mean values of casein-specific IgE antibodies and lower mean values of whey protein (BLG and ALA) - specific IgE antibodies compared with those less than 3 years of age. A single dominant allergenic milk protein was not identified within either of the two age groups, but the con centrations of the casein-specific IgE antibodies in children with more than 3 years of age tended to be higher than those of whey protein-specific IgE antibodies. CONCLUSION: Although statistically insignificant, the concentrations of the casein-specific IgE antibodies were higher in the group of more than 3 years of age than in the younger group. Moreover, the concentrations of the casein-specific IgE antibodies in children more than 3 years of age tended to be higher than those of whey proteins. These findings implicate that casein plays a certain role in the persistence of CMA.