Alpha Casein Research Articles

MILK ALLERGY IN JERSEY COWS: FIRST REPORT IN BRAZIL

Allergy to milk is the only auto-allergic disease in cattle. It is characterized by the retention and absorption of milk itself, making milk proteins, especially alpha casein, have access to the bloodstream, resulting in a type I hypersensitivity. The purpose of this report is to describe a case of auto-allergic disease in cattle. Here we report the case of a four-year-old Jersey female bovine, which was pregnant and weighing 400Kg. The animal was being treated at (hidden for revision) and had a history of an acute allergic reaction. Its owner reported sudden onset of skin lesions, salivation, and difficulty breathing. As the animal was participating in an agricultural exhibition, it was not being milked to generate mammary gland engorgement (milk retention). In physical examination findings wherein there is presence of multifocal urticarial papules and plaques, mainly on the sides and right and left flanks, besides the absence of pruritus, edema in mucocutaneous regions of the upper and lower eyelids of the right and left eyes, submandibular edema, excessive salivation, inspiratory dyspnea with half-open mouth breathing, respiratory distress, apathy, cough, tachycardia tachypnea, ruminal hypomotility and increased volume of the mammary gland, characterizing milk retention. Clinical suspicion was milk allergy with anaphylactic reaction (type I hypersensitivity). Treatment was based on promethazine administration (1mg/kg; IV) and immediate full milking of the mammary gland. After 12 h of treatment, clinical respiratory signs resolved, and after 24 h, skin changes resolved completely. Based on clinical signs and treatment success, the clinical suspicion of milk allergy causing anaphylactic reaction (type I hypersensitivity) was confirmed.

Investigation of the behavior of alpha casein upon binding to fluvastatin and pitavastatin: A spectroscopic study and molecular modeling

The interaction between alpha casein (α-CN) and two drugs, fluvastatin (FLU) and pitavastatin (PIT) was investigated using fluorescence, UV absorption and FTIR. In addition, the binding site was established by applying molecular modeling technique. Fluorescence data suggested that FLU and PIT quench the intrinsic fluorescence of α-CN. The binding constants for the interaction of FLU and PIT with α-CN were found to be (8.18±0.08)×104 M-1 and (9.04±0.07)×104 M-1, respectively, indicating that the binding affinity of PIT to α-CN was higher than that for FLU. The number of binding site FLU and PIT per α-CN were 1.06 and 1.04 respectively. Docking calculation showed the probable binding sites of FLU and PIT are located in the hydrophobic core of α-CN where the FLU and PIT are lined by hydrophobic residues and make three and four hydrogen bonds with FLU and PIT respectively. Simulation, molecular docking and experimental data reciprocally supported each other. Therefore, it can be concluded that α-CN can act as a carrier of FLU and PIT drugs.

Role of Lactium™ in Psychodermatology: The CERTAIN Trial# on Patients with Acne Vulgaris.

Stress plays an important role in the causation and aggravation of psychodermatological conditions such as acne vulgaris. Alpha casein hydrolysate (αs1-casein hydrolysate; Lactium) has been shown to decrease serum cortisol levels, reduce stress-related symptoms, and promote relaxation. “This study aimed to compare the efficacy and safety of Lactium™ plus standard care to those of standard of care alone in reducing stress levels and acne severity in patients with acne vulgaris.” The C.E.R.T.A.I.N trial (Name registered with Clinical Trials Registry-India-No. CTRI/2019/01/017172) is a randomized, controlled, multicenter, open-label, two-arm, investigator-initiated clinical trial. A total of 100 patients with moderate-to-severe acne vulgaris were enrolled and randomly assigned to one of the two groups: Lactium™ plus standard care or standard care alone. Stress levels were assessed using serum cortisol levels, Investigator's Global Assessment (IGA) acne severity scale scores, Perceived Stress Scale (PSS) scores, and the Hamilton Anxiety Rating Scale (HAM-A) scores. The Dermatology Life Quality Index (DLQI) was also used to assess the impact of the skin disease on patients' quality of life. At 12 weeks, stress levels were significantly lower in group A (Nixiyax plus standard of care) than that in group B(only standard care), as measured by the change in serum cortisol levels (4.75 ± 4.46 vs. −0.24 ± 5.22). Furthermore, the mean change in PSS scores (3.09 ± 2.04 vs. 0.90 ± 2.76) and HAM-A scores (5.11 ± 1.94 vs. 1.25 ± 3.13) was significant. Patients in both arms had a significant decrease in total, inflammatory, and noninflammatory acne lesions, as well as a significant improvement in DLQI and IGA scores. In patients with moderate-to-severe acne vulgaris, Lactium™ was found to be both safe and well-tolerated. Lactium™ plus standard care is more effective than standard care alone in reducing acne severity through stress reduction.

Using recombinant adhesive proteins as durable and green flame-retardant coatings

Current fire retardants are known to be toxic to humans and our environment. As environmental-friendly flame retardants (FRs), protein-based flame retardants have been studied extensively recently, even though they are not durable. In this study, we designed, synthesized and tested a durable protein-based FR through the fusion of the adhesion domain from either mussel foot protein-5 (mfp-5) or cellulose-binding domain (CBD) with flame retardant protein (SR protein and alpha casein). We first verified the expression of the recombinant proteins in Escherichia coli using Western blot. Then, we coated the fusion protein (carrying cell lysates) to cotton fabrics and wood and verified with Infrared (IR) spectroscopy. Using a vertical burning test and wood flammability test, we confirmed the flame retardancy of the materials after the protein coating. In the vertical burning test, the SR protein and alpha casein flame retardant proteins with the CBD adhesion domain showed a 50.0% and 43.3% increase in flame retardancy. The data is also consistent in the wood flame retardancy test. Confocal imaging experiments also suggested these new fire retardants can be preserved on the materials well even after washing. Overall, our results showed that flame-retardant proteins with adhesion domains are high potential candidates of green alternative flame retardants.

Valoración de las fracciones de caseínas en leche de cabra Criolla y Saanen

The objective of the present work was to compare the values of the percentages of the different fractions in the milk casein of Criollo and Saanen goats. For this purpose, 6 adult goats of each breed from a commercial goat herd, located in the semi-arid region of central western Argentina, were evaluated. The goats were fed on alfalfa and corn grain, and service was performed by natural mating. Samples were collected by hand milking, taking 100 c.c./animal at 15, 30 and 45 days post-partum. To determine the percentage of alpha, beta and kappa casein, the UREA-PAGE vertical gel electrophoresis method was used and the bands were read applying the semi-quantification method with ImageJ software. The results found showed that in both breeds the values of alpha casein were increasing, while those of beta and kappa casein decreased throughout the sampling. Likewise, it was found that alpha casein was higher in Saanen goat milk, while the percentages of beta and kappa casein were higher in Criollo goat milk, finding a significant statistical difference for alpha and beta casein atthe three times evaluated and in the case of kappa casein only in the 45-day sampling. It was concluded that Criollo goat milk has a lower percentage of alpha casein than Saanen goat milk, which would make it less allergenic and has a higher percentage of beta and kappa casein, a situation that would allow a quick coagulation for cheese production.and a higher stability and firmness in yogurts.

The Two Peptides Fragments of Goat Milk CSN1S2 Protein Blocked Insulin Receptor to Interact Ligand: In Silico Study

Insulin is a hormone that plays important role for tissue development and glucose homeostasis, it has significant implications for many chronic diseases, especially diabetes mellitus (DM). Alpha casein S2 (CSN1S2) isolated from Ethawah goat’s milk has eight peptides residue contain seven to twelve amino acid residues and has multiple functions, such as antimicrobial, immunomodulatory, and anti-oxidant. The purpose of this study is to observe the interaction between CSN1S2 protein peptides and Insulin Receptor. 3-D structure of Insulin Receptor is done by accessing RCSB PDB (ID: 4ZXB), protein was prepared using Discovery Studio BIOVIA 2019 and ligands were prepared using PyRx. Insulin receptor was docked to CSN1S2 protein peptides using HEX 8.0 software and visualized using Discovery Studio BIOVIA 2019. The results of this study showed different binding patterns, most of the chemistry bond were resulted between protein and ligands interaction are hydrogen bonds and electrostatic, the binding energy of CSN1S2 fragment 41-47 and fragment 214-221 to insulin receptor are -186.9 kJ/mol and -172.7 kJ/mol respectively. The caprine CSN1S2 protein peptides have ability to be used as therapeutic agents, such as DM because they can bind to specific sites of the insulin receptor and may reduce the insulin resistance mechanism.

Isolation and Molecular Characterization of Local Goat Milk Casein for Nutraceutical Value

Bioactive peptide from casein play a very important role in biological functionalities such as antioxidant and antimicrobial activities. Casein is the main protein that derived from goat milk which consists of alpha (α), beta (β) and kappa (κ) casein. Dietary protein such as casein from animal can provide rich source of bioactive peptide. However, the macromolecular protein such as cow milk can cause allergic response to certain individuals. On the other hand, goat milk have been known for its hypoallergenic and therapeutic properties in human nutrition and health. The purpose of this study is to extract casein from local breed goat milk and identify the molecular characterization of casein for nutraceutical value. The casein was successfully extracted using extraction method. Extraction is a common technique used to separate a desired substance when it is mixed with other components. The average percentage of casein obtained was 24.25%. Then, the casein was analysed by running it in the SDS-Page. The major fraction is β-casein and the minor is α-casein that can be seen between 20kDa and 30kDa respectively. There is no contaminated protein appear in the purified α-amylase. The result obtained in this study indicates that isolated casein from Malaysian goat milk was pure and can be used as bioactive peptide for nutraceutical value.

Chemical-Vapor-Assisted Electrospray Ionization for Increasing Analyte Signals in Electrospray Ionization Mass Spectrometry

We report a chemical-vapor-assisted electrospray ionization (ESI) technique to improve the detection sensitivity of ESI mass spectrometry (MS). This simple technique involves introducing a chemical vapor into the sheath gas around the nano-ESI spray tip or through a tubing with its outlet placed close to the spray tip. A variety of chemical vapors were tested and found to have varying degrees of effects on analyte signal intensities. The use of benzyl alcohol vapors in ESI was found to increase signal intensities of standard peptides by up to 4-fold. When this technique was combined with capillary liquid chromatography tandem MS (LC-MS/MS), the number of unique peptides identified in the acid hydrolysate of alpha casein increased by 45% and the number of peptides and proteins identified in a tryptic digest of E. coli cell lysate increased by 13% and 14%, respectively, along with an increased average match score. This technique could also increase the analyte signals for some small molecules, such as phenylephrine, by up to 3-fold. The increased analyte signals observed in the chemical-vapor-assisted ESI process is related to the enhancement of the ionization efficiency in ESI. The method can be readily implemented to an existing ESI mass spectrometer at minimum cost for improving detection sensitivity.

Cost-effective isobaric tagging for quantitative phosphoproteomics using DiART reagents

We describe the use of an isobaric tagging reagent, Deuterium isobaric Amine Reactive Tag (DiART), for quantitative phosphoproteomic experiments. Using DiART tagged custom mixtures of two phosphorylated peptides from alpha casein and their non-phosphorylated counterparts, we demonstrate the compatibility of DiART with TiO2 affinity purification of phosphorylated peptides. Comparison of theoretical vs. experimental reporter ion ratios reveals accurate quantification of phosphorylated peptides over a dynamic range of more than 15-fold. Using DiART labelling and TiO2 enrichment (DiART-TiO2) with large quantities of proteins (8 mg) from the cell lysate of model fungus Aspergillus nidulans, we quantified 744 unique phosphopeptides. Overlap of median values of TiO2 enriched phosphopeptides with theoretical values indicates accurate trends. Altogether these findings confirm the feasibility of performing quantitative phosphoproteomic experiments in a cost-effective manner using isobaric tagging reagents, DiART.

Investigating the deep supercooling ability of an Alaskan beetle, Cucujus clavipes puniceus, via high throughput proteomics

Cucujus clavipes puniceus is a freeze avoiding beetle capable of surviving the long, extremely cold winters of the Interior of Alaska. Previous studies showed that some individuals typically supercool to mean values of approximately -40 °C, with some individuals supercooling to as low as -58 °C, but these non-deep supercooling (NDSC) individuals eventually freeze if temperatures drop below this. However, other larvae, especially if exposed to very cold temperatures, supercool even further. These deep supercooling (DSC) individuals do not freeze even if cooled to -100 °C. In addition, the body water of the DSC larvae vitrifies (turns to a glass) at glass transition temperatures of -58 to -70 °C. This study examines the proteomes of DSC and NDSC larvae to assess proteins that may contribute to or inhibit the DSC trait. Using high throughput proteomics, we identified 138 proteins and 513 Gene Ontology categories in the DSC group and 104 proteins and 573 GO categories in the NDSC group. GO categories enriched in DSC include alcohol metabolic process, cellular component morphogenesis, monosaccharide metabolic process, regulation of biological quality, extracellular region, structural molecule activity, and antioxidant activity. Proteins unique to DSC include alpha casein precursor, alpha-actinin, vimentin, tropomyosin, beta-lactoglobulin, immunoglobulins, tubulin, cuticle proteins and endothelins.

Establishment and Characterization of Gene Expression of Two New Ovine Mammary Cell Lines: Preliminary Results.

Some post-translational modifications are necessary for the production of biopharmaceutical proteins, with a good specific action and a high biological activity. These modifications are obtained only by bioreactors based on eukaryotic cell as mammary cells. Bioreactors in vitro, with this kind of cell have been used for a viable and strategic production of biologically active recombinant proteins. For this reason, the establishment of a new line of mammary cells with high milk protein expression is an essential work. The main objectives of this study was to compare two methods, enzymatic and non-enzymatic, to establish ovine mammary cells culture and verify their gene expression of milk proteins such as beta lactoglobulin, alpha casein, beta casein and kappa casein with different treatments: LOS (lactating ovine serum) or FBS (fetal bovine serum) added to the culture medium, in the presence or absence of Matrigel. In this manner, an in vitro study was performed and the culture of two lines were established, digested (DL) and non-digested (NDL), of ovine mammary cell until the passage 12 (P12). In DL was observed just one cellular type that was positive for staining with vimentin. This cell line expressed beta lactoglobulin and beta casein genes with the FBS treatment and without Matrigel. The gene expression was lower (P=0.001) when compared to the NDL under the same conditions of culture. Then, the NDL expressed beta lactoglobulin, beta casein and kappa casein genes when treated with FBS without Matrigel. The treatment with LOS in the culture medium increased the gene expression of beta lactoglobulin for both cell lines. The growth curve was determined with both cell lines in P12 with FBS or LOS treatment. For the NDL, the type of medium had effect on the cell growth speed and was highest with the FBS treatment (P<0.05). However, the medium did not have effect on growth speed of LD (P>0.05) and no difference was observed at the NDL treated with LOS (P>0.05). The NDL was positive for staining with vimentin and cytokeratin. In conclusion, the in vitro culture of NDL and DL was established up to the P12 with expression of milk protein and the LOS treatment increased the expression of beta lactoglobulin. The two cell lines culture were positive for staining of vimentina but only NDL was positive for cytokeratin. Research supported by FAPESP (2008/56138-2) and CNPq. (poster)

Effects of Surface Passivation on Gliding Motility Assays

In this study, we report differences in the observed gliding speed of microtubules dependent on the choice of bovine casein used as a surface passivator. We observed differences in both speed and support of microtubules in each of the assays. Whole casein, comprised of αs1, αs2, β, and κ casein, supported motility and averaged speeds of 966±7 nm/s. Alpha casein can be purchased as a combination of αs1 and αs2 and supported gliding motility and average speeds of 949±4 nm/s. Beta casein did not support motility very well and averaged speeds of 870±30 nm/s. Kappa casein supported motility very poorly and we were unable to obtain an average speed. Finally, we observed that mixing alpha, beta, and kappa casein with the proportions found in bovine whole casein supported motility and averaged speeds of 966±6 nm/s.

Effects of surface passivation on gliding motility assays

AbstractIn this study, we report differences in the observed gliding speed of microtubules dependent on the choice of bovine casein used as a surface passivator. We observed differences in both speed and support of microtubules in each of the assays. Whole casein, comprised of [alpha]~s1~, [alpha]~s2~, [beta], and [kappa] casein, supported motility and averaged speeds of 966 ± 7 nm/s. Alpha casein can be purchased as a combination of s1 and s2 and supported gliding motility and average speeds of 949 ± 4 nm/s. Beta casein did not support motility very well and averaged speeds of 870 ± 30 nm/s. Kappa casein supported motility very poorly and we were unable to obtain an average speed. Finally, we observed that mixing alpha, beta, and kappa casein with the proportions found in bovine whole casein supported motility and averaged speeds of 966 ± 7 nm/s.

Insulin autoantibodies with high affinity to the bovine milk protein alpha casein

Insulin autoantibodies (IAA) can appear in children within months of introducing solid foods to the diet and before clinical type 1 diabetes. The aim of this study was to determine whether infant dietary antigens could be immunizing agents of IAA. To this end, IAA binding to [(125) I]insulin was competed with food preparations and extracts of foods encountered in the infant diet (milk formulas, bovine milk, wheat flour, fowl meal). Bovine milk powder extracts inhibited IAA-positive samples from six of 53 children (age 0·3-14·0 years) participating in German prospective cohorts. Inhibition in these sera ranged from 23 to 100%. Competition was abolished when hydrolyzed milk powder was used. Competition with protein components of bovine milk found that two of the six milk-reactive sera were inhibited strongly by alpha- and beta-casein; none were inhibited by the milk proteins bovine serum albumin or lactoglobulins. The two casein-reactive sera had high affinity to alpha-casein (1·7×10(9) ; 3·1×10(9) l/mol), and lesser affinity to beta-casein (4·0×10(8) ; 7·0×10(7) l/mol) and insulin (2·6×10(8) ; 1·6×10(8) l/mol). No children with milk-reactive IAA developed autoantibodies to other islet autoantigens or diabetes (median follow-up 9·8 years). These results suggest that autoimmunity to insulin can occur infrequently via cross-reactivity to food proteins, but this form of IAA immunization does not appear to be associated with progression to diabetes.

In Vitro and In Vivo Effects of Natural Putative Secretagogues of Glucagon-Like Peptide-1 (GLP-1)

Glucagon-like peptide-1 (GLP-1) is an intestinal hormone with well-established glucose-lowering activity. The in vitro and in vivo actions of natural putative secretagogues of GLP-1 were investigated. The acute GLP-1 releasing activity of olive leaf extract (OLE), glutamine (GLN), alpha casein (ACAS), beta casein (BCAS) and chlorogenic acid (CGA) were assessed in STC-1 cells and C57BL/6 mice. All compounds except ACAS significantly increased acute in vitro GLP-1 secretion (66–386%; P<0.05–0.001). Oral gavage of OLE and GLN modestly increased plasma GLP-1 concentrations (48% and 41%, respectively), but did not lower glycaemic excursions. OLE and GLN are potent stimulators of GLP-1 secretion both in vitro and in vivo and chronic studies should assess their suitability as nutritional therapies for type 2 diabetes.

Alpha casein micelles show not only molecular chaperone-like aggregation inhibition properties but also protein refolding activity from the denatured state

Casein micelles are a major component of milk proteins. It is well known that casein micelles show chaperone-like activity such as inhibition of protein aggregation and stabilization of proteins. In this study, it was revealed that casein micelles also possess a high refolding activity for denatured proteins. A buffer containing caseins exhibited higher refolding activity for denatured bovine carbonic anhydrase than buffers including other proteins. In particular, a buffer containing α-casein showed about a twofold higher refolding activity compared with absence of α-casein. Casein properties of surface hydrophobicity, a flexible structure and assembly formation are thought to contribute to this high refolding activity. Our results indicate that casein micelles stabilize milk proteins by both chaperone-like activity and refolding properties.

Effects of surface passivation on gliding motility assays

AbstractIn this study, we report differences in the observed gliding speed of microtubules dependent on the choice of bovine casein used as a surface passivator. We observed differences in both speed and support of microtubules in each of the assays. Whole casein, comprised of [alpha]~s1~, [alpha]~s2~, [beta], and [kappa] casein, supported motility and averaged speeds of966 ± 7 nm/s. Alpha casein can be purchased as a combination of s1 and s2 and supported gliding motility and average speeds of 949 ± 4 nm/s. Beta casein did not support motility very well and averaged speeds of 870 ± 30 nm/s. Kappa casein supported motility very poorly and we were unable to obtain an average speed. Finally,we observed that mixing alpha, beta, and kappa casein with the proportions found in bovine whole casein supported motility and averaged speeds of 966 ± 7 nm/s.

Efeito da gordura protegida sobre a produção e composição do leite em ovelhas da raça Bergamácia

Objetivou-se avaliar o efeito da gordura protegida sobre a produção e composição do leite em ovelhas da raça Bergamácia. Utilizaram-se 77 ovelhas distribuídas, por ordem de parição e idade, em duas dietas: uma composta de silagem de milho e concentrado; e outra com gordura protegida (35 g/ovelha/dia) no concentrado. As dietas foram isoenergéticas e isoproteicas e continham 70% NDT e 16% PB com base na matéria seca. Os cordeiros permaneceram com suas mães até os 45 dias de idade, quando foram desmamados. Quarenta e oito horas após o parto, deu-se início a ordenha realizada uma vez ao dia, às 7 h, para mensuração diária da produção de leite durante 60 dias. Amostras de leite foram coletadas semanalmente para análise da composição centesimal e do teor de caseína. A dieta com gordura protegida promoveu maior produção média diária de leite após a desmama (0,531 vs 0,489 kg/dia), entretanto, a produção total do período não diferiu entre controle e com gordura protegida (0,453 vs 0,468 kg/dia, respectivamente). Não foi observada influência das dietas sobre os teores de gordura do leite até a sexta semana de lactação. As diferenças ocorreram apenas após a desmama. Os teores de alfa e beta caseínas diferiram entre os grupos controle e gordura protegida (31,73 vs 18,56 Kda para alfa-caseína e 32,67 vs 26,44 Kda para beta-caseína, respectivamente). A adição de gordura protegida na dieta não altera a produção nem a composição centesimal do leite até a desmama.

Mechanism of immunopotentiation by aluminum-containing adjuvants elucidated by the relationship between antigen retention at the inoculation site and the immune response

The relationship between depot formation and immunopotentiation was studied by comparing the retention of antigen at the inoculation site with antibody production in rats. A model 111In-labeled alpha casein (IDCAS) antigen was formulated into four vaccines: IDCAS adsorbed onto either aluminum hydroxide adjuvant (AH) or aluminum phosphate adjuvant (AP); non-adsorbed IDCAS with phosphate-treated AP (PTAP); and IDCAS solution. Gamma scintigraphy showed the order of retention following subcutaneous administration to be: AH adsorbed > AP adsorbed > non-adsorbed with PTAP = solution. The antibody titers followed the order: non-adsorbed with PTAP = AP adsorbed > AH adsorbed ≫ solution. The presence of an aluminum-containing adjuvant was essential for immunopotentiation, but retention of the antigen at the inoculation site was not required.

Production of Synthetic Methionine-Free and Synthetic Methionine-Limited Alpha Casein: Protein Foodstuff for Patients with Homocystinuria due to Cystathionine Beta-Synthase Deficiency

The aim of this study was to evaluate the possibility that synthetic forms of methionine-free alpha-casein and methionine-limited alpha casein could be produced by recombinant means to form the basis for developing an industrial-scale process for the provision of a foodstuff suitable for patients with homocystinuria due to cystathionine beta-synthase (CBS) deficiency. As a first step, two forms of alpha casein gene, encoding methionine-free alpha casein (Fcas) or a methionine-limited alpha casein (Mcas), were synthesised and expressed in Escherichia coli. Using the overexpression vector pET28a, both genes were highly expressed in E. coli in soluble form as well as in inclusion bodies. The two recombinant proteins were purified by the one step methods using the fused His-tag and the Ni(2+)column and validated by Western blot analysis. This work paves the way for industrial-scale production of proteins suitable for patients with homocystinuria due to CBS deficiency.