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.
Crystallization ; Lactoglobulins

No abstract available.
Immunoglobulins* ; Lactoglobulins* ; Milk*

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.
Amino Acids ; Bandages ; Lactoglobulins

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.
Animals ; Animals, Genetically Modified ; genetics ; Female ; Fibroblasts ; Gene Knock-In Techniques ; Gene Knockout Techniques ; Goats ; genetics ; Humans ; Lactoferrin ; genetics ; Lactoglobulins ; genetics ; Milk ; chemistry ; Nuclear Transfer Techniques ; Plasmids ; Pregnancy ; Transfection

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.
Antibodies ; Caseins ; Child* ; Humans ; Immunoglobulin E* ; Lactoglobulins ; Milk Hypersensitivity* ; Milk Proteins ; Milk* ; Whey Proteins

No abstract available.
Child* ; Humans ; Immunoglobulins ; Infant, Newborn* ; Lactoglobulins ; Milk Proteins* ; Milk* ; Mothers* ; Postpartum Period*

PURPOSE: It is difficult to detect small amount of aspiration into the lungs due to the lack of safe, sensitive and specific diagnostic tool. Recently, in animal studies, it has been reported that immunocytochemistry for lactoglobulin can be used to detect the minimal aspiration of cow milk. So, we tried to determine the difference between immunocytochemistry for lactoglobulin and Oil Red O stain of alveolar macrophages in cow milk aspirated mice. METHODS: Fifty seven mice with 6-8 weeks old and 30-40 g weighing were used. Mice received either single or multiple intranasal instillation of 0.05 ml cow milk for study and saline for control under the anesthesia with ketamine and xylazine. The trachea of mouse was cannulated with 20G Jelco needle and then, mouse lungs were lavaged 3 times with 0.5 ml of phosphate buffer solution at 4 hours, 12 hours, 24 hours after the last milk or saline instillation. Cells in bronchoalveolar lavage fluid(BALF) were stained with Oil Red O and immunocytochemistry for beta-lactoglobulin. RESULTS: After single aspiration of milk, no cellular difference was found in bronchoalveolar lavage fluid(BALF) when compared with saline aspirated group at 4 hours. But after repeated aspiration of milk, significant change was observed in the number of alveolar macrophage, neutrophil, lymphocyte and eosinophil. Immunocytochemical reactivity was not observed in alveolar macrophages of saline aspirated group. Lipid-laden alveolar macrophages were recovered rarely in Oil Red O staining. Immunocytochemical staining displayed stain-positive alveolar macrophages for beta-lactoglobulin at 4 hours after milk aspiration, it had a peak at 12 hours and decreased markedly at 24 hours. Immunocytochemical stain positive alveolar macrophages appeared similarly in number between single and repeated aspiration group. CONCLUSION: These observations suggested that alveolar macrophages could be detected more easily on immunocytochemistry for lactoglobulin than Oil Red O stain and immunocytochemistry could be used as a sensitive & specific diagnostic method for the detection of milk aspiration.
Anesthesia ; Animals ; Bronchoalveolar Lavage ; Eosinophils ; Immunohistochemistry ; Ketamine ; Lactoglobulins ; Lung ; Lymphocytes ; Macrophages, Alveolar* ; Mice* ; Milk* ; Needles ; Neutrophils ; Trachea ; Xylazine

OBJECTIVETo 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.

METHODSThe 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.

RESULTSRenaturation of recombinant bovine β-lactoglobulin resulted in a more compact structure resembling the natural counterpart with stronger IgE-binding capacity.

CONCLUSIONThe 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.

Allergens ; Animals ; Cattle ; Circular Dichroism ; Enzyme-Linked Immunosorbent Assay ; Immunoglobulin E ; Lactoglobulins ; chemistry ; metabolism ; Models, Molecular ; Protein Binding ; Protein Conformation ; Protein Denaturation ; Protein Folding ; Spectrometry, Fluorescence ; methods

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.
Allergens ; Caseins ; Complex Mixtures ; Electrophoresis ; Heating ; Hot Temperature* ; Humans ; Hydrolysis* ; Immunoblotting ; Immunoglobulin E ; Lactalbumin ; Lactoglobulins ; Milk ; Milk Hypersensitivity ; Milk Proteins* ; Molecular Weight ; Sodium ; Water

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.
Allergens ; Asthma ; Caseins ; Dermatitis, Atopic ; Diagnosis ; Follow-Up Studies ; Humans ; Hypersensitivity ; Immunoglobulin E* ; Infant ; Infant, Newborn* ; Lactoglobulins ; Milk Hypersensitivity ; Milk* ; Parents ; Physical Examination