Genetic variability among and within domestic Old and New World camels at the α-lactalbumin gene (LALBA) reveals new alleles and polymorphisms responsible for differential expression.

α-Lactalbumin (α-LA), which is encoded by the LALBA gene, is a major whey protein that binds to Ca2+ and facilitates lactose synthesis as a regulatory subunit of the synthase enzyme complex. In addition, it has been shown to play central roles in immune modulation, cell-growth regulation, and antimicrobial activity. In this study, a multitechnical approach was used to fully characterize the LALBA gene and its variants in both coding and regulatory regions for domestic camelids (dromedary, Bactrian camel, alpaca, and llama). The gene analysis revealed a conserved structure among the camelids, but a slight difference in size (2,012 bp on average) due to intronic variations. Promoters were characterized for the transcription factor binding sites (11 found in total). Intraspecies sequence comparison showed 36 SNPs in total (2 in the dromedary, none in the Bactrian camel, 22 in the alpaca, and 12 in the llama), whereas interspecies comparison showed 86 additional polymorphic sites. Eight SNPs were identified as trans-specific polymorphisms, and 2 of them (g.112A>G and g.1229A>G) were particularly interesting in the New World camels. The first creates a new binding site for transcription factor SP1. An enhancing effect of the g.112G variant on the expression was demonstrated by 3 independent pGL3 gene reporter assays. The latter is responsible for the p.78Ile>Val AA replacement and represents novel allelic variants (named LALBA A and B). A link to protein variants has been established by isoelectric focusing (IEF), and bioinformatics analysis revealed that carriers of valine (g.1229G) have a higher glycosylation rate. Genotyping methods based on restriction fragment length polymorphism (PCR-RFLP) were set up for both SNPs. Overall, adenine was more frequent (0.54 and 0.76) at both loci. Four haplotypes were found, and the AA and GA were the most common with a frequency of 0.403 and 0.365, respectively. Conversely, a putative biological gain characterizes the haplotype GG. Therefore, opportunities for rapid directional selection can be realized if this haplotype is associated with favorable milk protein properties. This study adds knowledge at the gene and protein level for α-LA (LALBA) in camelids and importantly contributes to a relatively unexplored research area in these species.

Whey protein dynamics in goat mammary secretions during colostrum and early lactation periods.

The protein composition in goat milk undergoes changes throughout the different lactation periods, displaying distinct characteristics that are influenced by the dynamic nature of protein composition and concentration during the transition from colostrum secretion to mature milk. To evaluate the dynamics of whey proteins of Saanen goats during the colostral phase and the first month of lactation, 110 milk samples from 11 healthy mammary halves of seven Saanen goats were selected through a clinical evaluation. Whey was obtained by rennet coagulation of the mammary secretion. The biuret method determined total protein concentration, and their fractions were identified by 12% dodecyl sulfate-polyacrylamide gel electrophoresis. Maximum concentrations of all protein fractions were observed in the first 12 h of lactation, reducing throughout the study. Modification of the protein predominance was also observed. The transition from colostrum secretion to milk occurred 5 or 7 d postpartum.

HAMLET a human milk protein-lipid complex induces a pro-inflammatory phenotype of myeloid cells.

HAMLET is a protein-lipid complex with a specific and broad bactericidal and tumoricidal activity, that lacks cytotoxic activity against healthy cells. In this study, we show that HAMLET also has general immune-stimulatory effects on primary human monocyte-derived dendritic cells and macrophages (Mo-DC and Mo-M) and murine RAW264.7 macrophages. HAMLET, but not its components alpha-lactalbumin or oleic acid, induces mature CD14low/- CD83+ Mo-DC and M1-like CD14+ CD86++ Mo-M surface phenotypes. Concomitantly, inflammatory mediators, including IL-2, IL-6, IL-10, IL-12 and MIP-1α, were released in the supernatant of HAMLET-stimulated cells, indicating a mainly pro-inflammatory phenotype. The HAMLET-induced phenotype was mediated by calcium, NFκB and p38 MAPK signaling in Mo-DCs and calcium, NFκB and ERK signaling in Mo-M as inhibitors of these pathways almost completely blocked the induction of mature Mo-DCs and M1-like Mo-M. Compared to unstimulated Mo-DCs, HAMLET-stimulated Mo-DCs were more potent in inducing T cell proliferation and HAMLET-stimulated macrophages were more efficient in phagocytosis of Streptococcus pneumoniae in vitro. This indicates a functionally activated phenotype of HAMLET-stimulated DCs and macrophages. Combined, we propose that HAMLET has a two-fold anti-bacterial activity; one inducing direct cytotoxic activity, the other indirectly mediating elimination of bacteria by activation of immune cells of the myeloid lineage.

An innovative approach to polycystic ovary syndrome.

Myo-inositol and D-chiro-inositol are insulin sensitising agents. In the ovary, myo-inositol acts as second messenger of Follicle Stimulating Hormone (FSH). Both molecules were administered to Polycystic Ovary Syndrome (PCOS) women. The gynaecologist Vittorio Unfer was the first to give specific value to myo-inositol for the treatment of PCOS: this important innovation opened new ways of research to identify efficient therapies based on myo-inositol alone or with low doses of D-chiro-inositol. Significant successes were also gained using myo-inositol in treating male and female infertility. Unfer's researches allowed to identify "the D-Chiro-Inositol Paradox in the Ovary" and the best myo-inositol/D-chiro-inositol ratio (40:1) for the treatment of PCOS. Furthermore, his studies allowed to improve the inositol's efficacy using alpha-lactalbumin. As shown in this review, the main stages of Unfer's scientific career have been closely intertwined with important phases of the recent pharmacological research about the topic.

A Comparative Review of the Extrinsic and Intrinsic Factors Regulating Lactose Synthesis.

Milk is critical for the survival of all mammalian offspring, where its production by a mammary gland is also positively associated with its lactose concentration. A clearer understanding of the factors that regulate lactose synthesis stands to direct strategies for improving neonatal health while also highlighting opportunities to manipulate and improve milk production and composition. In this review we draw a cross-species comparison of the extra- and intramammary factors that regulate lactose synthesis, with a special focus on humans, dairy animals, and rodents. We outline the various factors known to influence lactose synthesis including diet, hormones, and substrate supply, as well as the intracellular molecular and genetic mechanisms. We also discuss the strengths and limitations of various in vivo and in vitro systems for the study of lactose synthesis, which remains an important research gap.

A Comparative Review of the Cell Biology, Biochemistry, and Genetics of Lactose Synthesis.

Lactose is the primary carbohydrate in the milk of most mammals and is unique in that it is only synthesized by epithelial cells in the mammary glands. Lactose is also essential for the development and nutrition of infants. Across species, the concentration of lactose in milk holds a strong positive correlation with overall milk volume. Additionally, there is a range of examples where the onset of lactose synthesis as well as the content of lactose in milk varies between species and throughout a lactation. Despite this diversity, the precursors, genes, proteins and ions that regulate lactose synthesis have not received the depth of study they likely deserve relative to the significance of this simple and abundant molecule. Through this review, our objective is to highlight the requirements for lactose synthesis at the biochemical, cellular and temporal levels through a comparative approach. This overview also serves as the prelude to a companion review describing the dietary, hormonal, molecular, and genetic factors that regulate lactose synthesis.

The human milk protein-lipid complex HAMLET disrupts glycolysis and induces death in Streptococcus pneumoniae.

HAMLET is a complex of human α-lactalbumin (ALA) and oleic acid and kills several Gram-positive bacteria by a mechanism that bears resemblance to apoptosis in eukaryotic cells. To identify HAMLET's bacterial targets, here we used Streptococcus pneumoniae as a model organism and employed a proteomic approach that identified several potential candidates. Two of these targets were the glycolytic enzymes fructose bisphosphate aldolase (FBPA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Treatment of pneumococci with HAMLET immediately inhibited their ATP and lactate production, suggesting that HAMLET inhibits glycolysis. This observation was supported by experiments with recombinant bacterial enzymes, along with biochemical and bacterial viability assays, indicating that HAMLET's activity is partially inhibited by high glucose-mediated stimulation of glycolysis but enhanced in the presence of the glycolysis inhibitor 2-deoxyglucose. Both HAMLET and ALA bound directly to each glycolytic enzyme in solution and solid-phase assays and effectively inhibited their enzymatic activities. In contrast, oleic acid alone had little to no inhibitory activity. However, ALA alone also exhibited no bactericidal activity and did not block glycolysis in whole cells, suggesting a role for the lipid moiety in the internalization of HAMLET into the bacterial cells to reach its target(s). This was verified by inhibition of enzyme activity in whole cells after HAMLET but not ALA exposure. The results of this study suggest that part of HAMLET's antibacterial activity relates to its ability to target and inhibit glycolytic enzymes, providing an example of a natural antimicrobial agent that specifically targets glycolysis.

Bisphenol A and benzophenone-3 exposure alters milk protein expression and its transcriptional regulation during functional differentiation of the mammary gland in vitro.

The plastic monomer and plasticizer bisphenol A (BPA), and the UV-filter benzophenone-3 (BP3) have been shown to have estrogenic activities that could alter mammary gland development. Our aim was to analyze whether BPA or BP3 direct exposure affects the functional differentiation of the mammary gland using an in vitro model. Mammary organoids were obtained and isolated from 8 week-old virgin female C57BL/6 mice and were differentiated on Matrigel with medium containing lactogenic hormones and exposed to: a) vehicle (0.01% ethanol); b) 1 × 10-9 M or 1 × 10-6 M BPA; or c) 1 × 10-12 M, 1 × 10-9 M or 1 × 10-6 M BP3 for 72 h. The mRNA and protein expression of estrogen receptor alpha (ESR1) and progesterone receptor (PR) were assessed. In addition, mRNA levels of PR-B isoform, glucocorticoid receptor (GR), prolactin receptor (PRLR) and Stat5a, and protein expression of pStat5a/b were evaluated at 72 h. The mRNA and protein expression of milk proteins and their DNA methylation status were also analyzed. Although mRNA level of PRLR and GR was similar between treatments, mRNA expression of ESR1, total PR, PR-B and Stat5a was increased in organoids exposed to 1 × 10-9 M BPA and 1 × 10-12 M BP3. Total PR expression was also increased with 1 × 10-6 M BPA. Nuclear ESR1 and PR expression was observed in all treated organoids; whereas nuclear pStat5a/b alveolar cells was observed only in organoids exposed to 1 × 10-9 M BPA and 1 × 10-12 M BP3. The beta-casein mRNA level was increased in both BPA concentrations and 1 × 10-12 M BP3, which was associated with hypomethylation of its promoter. The beta-casein protein expression was only increased with 1 × 10-9 M BPA or 1 × 10-12 M BP3. In contrast, BPA exposure decreased alpha-lactalbumin mRNA expression and increased DNA methylation level in different methylation-sensitive sites of the gene. Also, 1 × 10-9 M BPA decreased alpha-lactalbumin protein expression. Our results demonstrate that BPA or BP3 exposure alters milk protein synthesis and its transcriptional regulation during mammary gland differentiation in vitro.

Polymorphism association and expression analysis of alpha-lactalbumin (LALBA) gene during lactation in Nili Ravi buffalo.

Alpha-lactalbumin has been reported as a highly polymorphic gene that potentially alters the gene expression and is associated with milk composition in dairy breeds. Current study was conducted in two phases. In the first phase, polymorphisms identification in alpha-lactalbumin (LALBA) gene and its association with milk composition was performed. To identify the genetic polymorphism, Nili Ravi buffaloes at their second lactation were selected from Government livestock farm (Buffalo Research Institute, Pattoki). Genomic DNA was extracted from blood samples. After PCR amplification, products were sequenced, and data was analyzed. Results showed that the identified polymorphisms at chromosomal position 34310940 were found associated with major whey protein. In the second phase of study, milk samples were collected from five healthy mastitis-free Nili Ravi buffaloes in their second lactation for expression analysis of alpha-lactalbumin gene at their transition (day 15), mid (day 90), and late (day 250) lactation. Gene expression was observed highest in transition phase with a gradual decrease of expression in mid and late phase of lactation. Further studies are needed to explore the regulation of milk production genes and their translational efficiency during the course of lactation.

The dynamics of individual whey protein concentrations in cows' mammary secretions during the colostral and early lactation periods.

The bovine whey consists of more than 200 different types of proteins, of which ß-lactoglobulin, α-lactalbumin, serum albumin, immunoglobulins and lactoferrin predominate. However, their concentrations are not stable due to the existence of protein dynamics during a transition from colostrum secretion to mature milk. To evaluate the dynamics of whey proteins of Jersey cows during a colostral phase and first month of lactation and an influence of the number of lactations, 268 milk samples from 135 Jersey cows were selected through a clinical evaluation. Whey was obtained by rennet coagulation of the mammary secretion. The concentration of total proteins was determined by the biuret method and their fractions were identified by 12% dodecyl sulfate-polyacrylamide gel electrophoresis (12% SDS-PAGE). Maximum concentrations of all protein fractions were observed in the first 12 h of lactation, reducing over the course of the study. Modification of the protein predominance was also observed. The transition from colostrum secretion to milk occurred between 24 and 72 h postpartum. There was an influence of the number of lactations on the dynamics of whey proteins, indicating that multiparous cows had better immunological and nutritional quality when compared to primiparous cows.

Role of Charge and Hydrophobicity in Liprotide Formation: A Molecular Dynamics Study with Experimental Constraints.

Bovine α-lactalbumin (aLA) and oleate (OA) form a complex that has been intensively studied for its tumoricidal activity. Small-angle X-ray scattering (SAXS) has revealed that this complex consists of a lipid core surrounded by partially unfolded protein. We call this type of complex a liprotide. Little is known of the molecular interactions between OA and aLA, and no technique has so far provided any high-resolution structure of a liprotide. Here we have used coarse-grained (CG) molecular dynamics (MD) simulations, isothermal titration calorimetry (ITC) and SAXS to investigate the interactions between aLA and OA during the process of liprotide formation. With ITC we found that the strongest enthalpic interactions occurred at a molar ratio of 12.0±1.4:1 OA/aLA. Liprotides formed between OA and aLA at several OA/aLA ratios in silico were stable both in CG and in all-atom simulations. From the simulated structures we calculated SAXS spectra that show good agreement with experimentally measured patterns of matching liprotides. The simulations showed that aLA assumes a molten globular (MG) state, exposing several hydrophobic patches involved in interactions with OA. Initial binding of aLA to OA occurs in an area of aLA in which a high amount of positive charge is located, and only later do hydrophobic interactions become important. The results reveal how unfolding of aLA to expose hydrophobic residues is important for complex formation between aLA and OA. Our findings suggest a general mechanism for liprotide formation and might explain the ability of a large number of proteins to form liprotides with OA.

Role of selected amino acids on plasma IGF-I concentration in infants.

PURPOSE: Insulin-like growth factor-I (IGF-I) is related to growth and its secretion is modified by protein intake in early infancy. We examined the relationship of dietary protein and circulating amino acids on plasma IGF-I levels and early growth. METHODS: Healthy formula-fed infants (n = 213) were randomly assigned to receive either a protein-reduced infant formula with alpha-lactalbumin-enriched whey and free tryptophan and phenylalanine (IF) or an isocaloric standard formula without free amino acids (CF) for the first 120 days of life. A group of breastfed (BF) infants was studied as a non-randomized reference cohort. Biochemical variables were measured shortly after birth (subpopulation) and at an age of 120 days. A path analysis was used to explore the relationship between IGF-I, insulin and amino acids. Results are derived from secondary analyses of a randomized controlled trial. RESULTS: Plasma concentrations of IGF-I at 120 days were significantly higher in IF than in CF infants [58.5 (15.0) vs. 53.7 (9.95) ng/mL; p = 0.020]. BF infants showed lower IGF-I concentrations of 41.6 (10.7) ng/mL. All amino acids but Thr and Cit had a more marked effect on insulin than on IGF-I level. Considering weight, sex and feeding group, Trp explained an equal percentage of variance of IGF-I and insulin (total R 2 12.5 % of IGF-I and 12.3 % of insulin), while branched-chain AA explained an up to twofold higher variance of insulin than IGF-I. Compared to CF, IF explained 18.9 % of the IGF-I level (p = 0.03), while for insulin no direct effect was detectable. CONCLUSION: Higher IGF-I concentrations and growth velocities in infants receiving protein-reduced IF indicate that the protein concentration of an infant formula alone does not control IGF-I levels and growth. Other components (e.g., selected amino acids) of infant formulae might control directly or indirectly via insulin influence IGF-I.

The targeted proteins in tumor cells treated with the α-lactalbumin-oleic acid complex examined by descriptive and quantitative liquid chromatography-tandem mass spectrometry.

An α-lactalbumin-oleic acid (α-LA-OA) complex has exhibited selective antitumor activity in animal models and clinical trials. Although apoptosis and autophagy are activated and the functions of several organelles are disrupted in response to α-LA-OA, the detailed antitumor mechanism remains unclear. In this study, we used a novel technique, isobaric tags for relative and absolute quantitation, to analyze the proteome of tumor cells treated with α-LA-OA. We identified 112 differentially expressed proteins: 95 were upregulated to satisfy the metabolism of tumor cells; 17 were downregulated and targets of α-LA-OA. According to the differentially expressed proteins, α-LA-OA exerted its antitumor activity by disrupting cytoskeleton stability and cell motility, and by inhibiting DNA, lipid, and ATP synthesis, leading to cellular stress and activation of programmed cell death. This study provides a systematic evaluation of the antitumor activity of α-LA-OA, identifying its interacting targets and establishing the theoretical basis of α-LA-OA for use in cancer therapy.

Variability of the caprine whey protein genes and their association with milk yield, composition and renneting properties in the Sarda breed. 1. The LALBA gene.

The 5' flanking region and 3' UTR of the caprine LALBA gene were analysed by SSCP and sequencing. A total of nine SNPs were detected: three in the promoter region, two were synonymous coding SNPs at exon-1, and four SNPs were in exon-4, within the 3'UTR. The nucleotide changes located in the promoter region (c.-358T>C, c.-163G>A, c.-121T>G) were genotyped by SSCP in 263 Sarda goats to evaluate their possible effect on milk yield, composition and renneting properties. We observed an effect of the three SNPs on milk yield and lactose content. Genotypes TT and CT at c.-358T>C (P A (P C and c.-121T>G were part of transcription factors binding sites, potentially involved in modulating the LALBA gene expression. The LALBA genotype affected renneting properties (P < 0.001), as heterozygotes c.-358CT and c.-163GA were characterised by delayed rennet coagulation time and curd firming time and the lowest value of curd firmness. The present investigation increases the panel of SNPs and adds new information about the effects of the caprine LALBA gene polymorphism.

Development of an in vitro digestive model for studying the peptide profile of breast milk.

Human milk is a highly valuable food for newborns and infants. Its protein fraction plays an important role for the development of the newborn. In the present study, an in vitro digestive model, developed for resembling closely the digestive system of an infant, was applied to human milk in order to identify and characterize the peptide profile. The peptide profile obtained after digestion was analyzed by µLC-LTQ-Orbitrap-MS. A total of 149 peptides from ß-casein, 30 peptides from α-lactalbumin, 26 peptides from αs1-casein, 24 peptides from κ-casein, 28 peptides from osteopontin, and 29 from lactoferrin was recovered. The identified peptide profile of partially hydrolyzed proteins, such as caseins, α-lactalbumin, and osteopontin, was different from that previously reported demonstrating a different performance of the developed neonatal digestive system with respect to other previously applied. These results would be useful as a starting point to investigate the physiological function of breast milk peptides.

Molecular mechanisms of the cytotoxicity of human α-lactalbumin made lethal to tumor cells (HAMLET) and other protein-oleic acid complexes.

Although HAMLET (human α-lactalbumin made lethal to tumor cells), a complex formed by human α-lactalbumin and oleic acid, has a unique apoptotic activity for the selective killing of tumor cells, the molecular mechanisms of expression of the HAMLET activity are not well understood. Therefore, we studied the molecular properties of HAMLET and its goat counterpart, GAMLET (goat α-lactalbumin made lethal to tumor cells), by pulse field gradient NMR and 920-MHz two-dimensional NMR techniques. We also examined the expression of HAMLET-like activities of complexes between oleic acid and other proteins that form a stable molten globule state. We observed that both HAMLET and GAMLET at pH 7.5 were heterogeneous, composed of the native protein, the monomeric molten globule-like state, and the oligomeric species. At pH 2.0 and 50 °C, HAMLET and GAMLET appeared in the monomeric state, and we identified the oleic acid-binding site in the complexes by two-dimensional NMR. Rather surprisingly, the binding site thus identified was markedly different between HAMLET and GAMLET. Furthermore, canine milk lysozyme, apo-myoglobin, and ß2-microglobulin all formed the HAMLET-like complex with the anti-tumor activity, when the protein was treated with oleic acid under conditions in which their molten globule states were stable. From these results, we conclude that the protein portion of HAMLET, GAMLET, and the other HAMLET-like protein-oleic acid complexes is not the origin of their cytotoxicity to tumor cells and that the protein portion of these complexes plays a role in the delivery of cytotoxic oleic acid molecules into tumor cells across the cell membrane.

Combination of sunitinib with anti-tumor vaccination inhibits T cell priming and requires careful scheduling to achieve productive immunotherapy.

Sunitinib, a protein tyrosine kinase inhibitor is the frontline therapy for renal and gastrointestinal cancers. We hypothesized that by virtue of its well documented tumor apoptosis and immune adjuvant properties, combination of Sunitinib with anti-tumor immunotherapeutics will provide synergistic inhibition of tumor growth. Our study was designed to evaluate the impact of Sunitinib on immunotherapy mediated anti-tumor immune responses and evaluate its efficacy as a combinatorial therapy with tumor targeted immunotherapeutic vaccination. Mice immunized with recombinant α-lactalbumin, a lactation protein expressed on majority of breast tumors were treated with 1 mg of Sunitinib for seven consecutive days beginning (1) concurrently, on the day of α-lactalbumin immunization or (2) sequentially, on day 9 after immunization. Ten-day lymph nodes or 21 day spleens were tested by ELISPOT assays and flow cytometry to evaluate responsiveness to α-lactalbumin immunization in presence of Sunitinib and distribution of cells involved in T cell antigen priming and proliferation in different lymphoid compartments. In addition, therapeutic efficacy of the α-lactalbumin/ Sunitinib combination was evaluated by monitoring tumor growth in the 4T1 transplanted tumor model. Our studies reveal that concurrent administration of Sunitinib with active vaccination against a targeted tumor antigen inhibits priming to the immunogen due to a drastic decrease in CD11b+CD11c+ antigen presenting cells, leading to failure of vaccination. However, sequential delivery of Sunitinib timed to avoid the priming phase of vaccination results in the desired vaccination mediated boost in immune responses.

Low-Protein Infant Formula Enriched with Alpha-Lactalbumin during Early Infancy May Reduce Insulin Resistance at 12 Months: A Follow-Up of a Randomized Controlled Trial.

High protein intake during infancy results in accelerated early weight gain and potentially later obesity. The aim of this follow-up study at 12 months was to evaluate if modified low-protein formulas fed during early infancy have long-term effects on growth and metabolism. In a double-blinded RCT, the ALFoNS study, 245 healthy-term infants received low-protein formulas with either alpha-lactalbumin-enriched whey (α-lac-EW; 1.75 g protein/100 kcal), casein glycomacropeptide-reduced whey (CGMP-RW; 1.76 g protein/100 kcal), or standard infant formula (SF; 2.2 g protein/100 kcal) between 2 and 6 months of age. Breastfed (BF) infants served as a reference. At 12 months, anthropometrics and dietary intake were assessed, and serum was analyzed for insulin, C-peptide, and insulin-like growth factor 1 (IGF-1). Weight gain between 6 and 12 months and BMI at 12 months were higher in the SF than in the BF infants (p = 0.019; p < 0.001, respectively), but were not significantly different between the low-protein formula groups and the BF group. S-insulin and C-peptide were higher in the SF than in the BF group (p < 0.001; p = 0.003, respectively), but more alike in the low-protein formula groups and the BF group. Serum IGF-1 at 12 months was similar in all study groups. Conclusion: Feeding modified low-protein formula during early infancy seems to reduce insulin resistance, resulting in more similar growth, serum insulin, and C-peptide concentrations to BF infants at 6-months post intervention. Feeding modified low-protein formula during early infancy results in more similar growth, serum insulin, and C-peptide concentrations to BF infants 6-months post intervention, probably due to reduced insulin resistance in the low-protein groups.

Interaction between a photoisomerizable azobenzene compound and alpha-lactalbumin: Spectroscopic and computational simulation studies.

The combination of light and photoresponsive compounds provides a peculiar way of regulating biological systems. Azobenzene is a classical organic compound with photoisomerization properties. Exploring the interactions between azobenzene and proteins can deepen the biochemical applications of the azobenzene compounds. In this paper, the interaction of 4-[(2,6-dimethylphenyl)diazenyl]-3,5-dimethylphenol with alpha-lactalbumin was investigated by UV-Vis absorption spectra, multiple fluorescence spectra, computer simulations, and circular dichroism spectra. Most critically, the interaction differences between proteins and the trans- and cis-isomer of ligands have been analyzed and compared. Results showed that both isomers of ligands were bound to alpha-lactalbumin to form ground state complexes and statically quenched the steady-state fluorescence of alpha-lactalbumin. The van der Waals forces and hydrogen bonding dominated the binding; the difference is that the binding of the cis-isomer to alpha-lactalbumin is more rapidly stabilized, and the binding strength is greater than the trans-isomer. These binding differences were modeled and analyzed by molecular docking and kinetic simulations, and we found that both isomers bind through the hydrophobic aromatic cluster 2 of alpha-lactalbumin. However, the bent structure of the cis-isomer is more closely aligned with the construction of the aromatic cluster and may have contributed to the above differences.

Evaluation of Children with Cow's Milk Allergy Who Received Measles or Measles, Mumps, and Rubella Vaccines Containing Alpha-Lactalbumin.

Objective: Cases of cow's milk allergy (CMA) who reacted to measles or measles, mumps, and rubella (MMR) vaccines containing alpha-lactalbumin have been reported. The purpose of this study was to assess patients with CMA who received measles or MMR vaccines containing alpha-lactalbumin, as well as the characteristics of those who developed reactions to these vaccines. Study Design: Patients followed up in the allergy clinic for CMA and who received measles or MMR vaccines containing alpha-lactalbumin at 9 or 12 months of age were included in the study, and their characteristics were analyzed retrospectively from the hospital registry system. Results: Forty-nine patients were included in the study. Six patients received the measles vaccine, whereas 43 patients received the MMR vaccine containing alpha-lactalbumin. Vaccine skin tests were performed on these 6 patients. One patient had a positive intradermal test, so an alternative vaccine not containing alpha-lactalbumin was administered. The other 5 patients were vaccinated, and no reaction was observed. Anaphylaxis was observed in 3 of 43 patients who received the MMR vaccine containing alpha-lactalbumin. In all of these patients, the first reaction to dairy products was anaphylaxis. In 2 of those patients, cow's milk-specific IgE (spIgE) levels were >100 kU/L, and alpha-lactalbumin-spIgE levels were also high at 97 and 90 kU/L. The third patient's cow's milk-spIgE level was 15.9 kU/L, whereas the alpha-lactalbumin-spIgE level was 0.04 kU/L. Conclusion: Especially in patients with an initial reaction of anaphylaxis to dairy products and high cow's milk-spIgE levels, the risk of reaction is high with the MMR vaccine.