Soy protein diet alters expression of hepatic genes regulating fatty acid and thyroid hormone metabolism in the male rat

USDA-ARS?s Scientific Manuscript database

We determined effects of soy protein (SPI) and the isoflavone genistein (GEN) on mRNA expression of key lipid metabolism and thyroid hormone system genes in young adult, male Sprague-Dawley rats. SPI-fed rats had less retroperitoneal fat and less hepato-steatosis than casein (CAS, control protein)-...

Substitution of soy protein for casein prevents oxidative modification and inflammatory response induced in rats fed high fructose diet.

PubMed

Sreeja, S; Geetha, Rajagopalan; Priyadarshini, Emayavaramban; Bhavani, Krishnamoorthy; Anuradha, Carani Venkatraman

2014-01-01

Fructose-rich diet is known to cause metabolic dysregulation, oxidative stress, and inflammation. We aimed to compare the effects of two dietary proteins of animal and plant origins on fructose-induced oxidative stress and inflammatory changes in liver. Wistar rats were fed either starch or fructose (60%) diet with casein or soy protein (20%) as the protein source for 8 weeks. Glucose and insulin, glycated hemoglobin and fructosamine, AOPP, and FRAP were determined in circulation. Intracellular ROS, oxidatively modified proteins (4-HNE and 3-NT adducts), adiponectin, TNF- α , IL-6 and PAI-1 mRNA expression, phosphorylation and activation of JNK and IKK β , and NF- κ B binding activity were assayed in liver. In comparison with starch fed group, fructose + casein group registered significant decline in antioxidant potential and increase in plasma glucose, insulin, and glycated proteins. Increased ROS production, 4-HNE and 3-NT modified proteins, JNK and IKK β activation, and NF- κ B binding activity were observed in them along with increased gene expression of PAI-1, IL-6, and TNF- α and decreased adiponectin expression. Substitution of soy protein for casein reduced oxidative modification and inflammatory changes in fructose-fed rats. These data suggest that soy protein but not casein can avert the adverse effects elicited by chronic consumption of fructose.

Genetic variability of the equine casein genes.

PubMed

Brinkmann, J; Jagannathan, V; Drögemüller, C; Rieder, S; Leeb, T; Thaller, G; Tetens, J

2016-07-01

The casein genes are known to be highly variable in typical dairy species, such as cattle and goat, but the knowledge about equine casein genes is limited. Nevertheless, mare milk production and consumption is gaining importance because of its high nutritive value, use in naturopathy, and hypoallergenic properties with respect to cow milk protein allergies. In the current study, the open reading frames of the 4 casein genes CSN1S1 (αS1-casein), CSN2 (β-casein), CSN1S2 (αS2-casein), and CSN3 (κ-casein) were resequenced in 253 horses of 14 breeds. The analysis revealed 21 nonsynonymous nucleotide exchanges, as well as 11 synonymous nucleotide exchanges, leading to a total of 31 putative protein isoforms predicted at the DNA level, 26 of which considered novel. Although the majority of the alleles need to be confirmed at the transcript and protein level, a preliminary nomenclature was established for the equine casein alleles. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Epigenetic Modifications Unlock the Milk Protein Gene Loci during Mouse Mammary Gland Development and Differentiation

PubMed Central

Rijnkels, Monique; Freeman-Zadrowski, Courtneay; Hernandez, Joseph; Potluri, Vani; Wang, Liguo; Li, Wei; Lemay, Danielle G.

2013-01-01

Background Unlike other tissues, development and differentiation of the mammary gland occur mostly after birth. The roles of systemic hormones and local growth factors important for this development and functional differentiation are well-studied. In other tissues, it has been shown that chromatin organization plays a key role in transcriptional regulation and underlies epigenetic regulation during development and differentiation. However, the role of chromatin organization in mammary gland development and differentiation is less well-defined. Here, we have studied the changes in chromatin organization at the milk protein gene loci (casein, whey acidic protein, and others) in the mouse mammary gland before and after functional differentiation. Methodology/Principal Findings Distal regulatory elements within the casein gene cluster and whey acidic protein gene region have an open chromatin organization after pubertal development, while proximal promoters only gain open-chromatin marks during pregnancy in conjunction with the major induction of their expression. In contrast, other milk protein genes, such as alpha-lactalbumin, already have an open chromatin organization in the mature virgin gland. Changes in chromatin organization in the casein gene cluster region that are present after puberty persisted after lactation has ceased, while the changes which occurred during pregnancy at the gene promoters were not maintained. In general, mammary gland expressed genes and their regulatory elements exhibit developmental stage- and tissue-specific chromatin organization. Conclusions/Significance A progressive gain of epigenetic marks indicative of open/active chromatin on genes marking functional differentiation accompanies the development of the mammary gland. These results support a model in which a chromatin organization is established during pubertal development that is then poised to respond to the systemic hormonal signals of pregnancy and lactation to achieve the full functional capacity of the mammary gland. PMID:23301053

Distinct roles of prolactin, epidermal growth factor, and glucocorticoids in β-casein secretion pathway in lactating mammary epithelial cells.

PubMed

Kobayashi, Ken; Oyama, Shoko; Kuki, Chinatsu; Tsugami, Yusaku; Matsunaga, Kota; Suzuki, Takahiro; Nishimura, Takanori

2017-01-15

Beta-casein is a secretory protein contained in milk. Mammary epithelial cells (MECs) synthesize and secrete β-casein during lactation. However, it remains unclear how the β-casein secretion pathway is developed after parturition. In this study, we focused on prolactin (PRL), epidermal growth factor (EGF), and glucocorticoids, which increase in blood plasma and milk around parturition. MECs cultured with PRL, EGF and dexamethasone (DEX: glucocorticoid analog) developed the β-casein secretion pathway. In the absence of PRL, MECs hardly expressed β-casein. EGF enhanced the expression and secretion of β-casein in the presence of PRL and DEX. DEX treatment rapidly increased secreted β-casein concurrent with enhancing β-casein expression. DEX also up-regulated the expression of SNARE proteins, such as SNAP-23, VAMP-8 and Syntaxin-12. Furthermore, PRL and DEX regulated the expression ratio of α s1 -, β- and κ-casein. These results indicate that PRL, EGF and glucocorticoids have distinct roles in the establishment of β-casein secretion pathway. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Casein Kinase I Isoform Hrr25 Is a Negative Regulator of Haa1 in the Weak Acid Stress Response Pathway in Saccharomyces cerevisiae

PubMed Central

Collins, Morgan E.; Black, Joshua J.

2017-01-01

ABSTRACT Haa1 is a transcription factor that adapts Saccharomyces cerevisiae cells to weak organic acid stresses by activating the expression of various genes. Many of these genes encode membrane proteins, such as TPO2 and YRO2. How Haa1 is activated by weak acids is not clear. Here, we show that casein kinase I isoform Hrr25 is an important negative regulator of Haa1. Haa1 is known to be multiply phosphorylated. We found that mutations in HRR25 lead to reduced Haa1 phosphorylation and increased expression of Haa1 target genes and that Hrr25 interacts with Haa1. The other three casein kinase I isoforms, Yck1, Yck2, and Yck3, do not seem to play critical roles in Haa1 regulation. Hrr25 has a 200-residue C-terminal region, including a proline- and glutamine-rich domain. Our data suggest that the C-terminal region of Hrr25 is required for normal inhibition of expression of Haa1 target genes TPO2 and YRO2 and is important for cell growth but is not required for cell morphogenesis. We propose that Hrr25 is an important regulator of cellular adaptation to weak acid stress by inhibiting Haa1 through phosphorylation. IMPORTANCE Our study has revealed the casein kinase I protein Hrr25 to be a negative regulator of Haa1, a transcription factor mediating the cellular response to stresses caused by weak acids. Many studies have focused on the target genes of Haa1 and their roles in weak acid stress responses, but little has been reported on the regulatory mechanism of Haa1. Weak acids, such as acetic acid, have long been used for food preservation by slowing down the growth of fungal species, including S. cerevisiae. In the biofuel industry, acetic acid in the lignocellulosic hydrolysates limits the production of ethanol, which is undesirable. By understanding how Haa1 is regulated, we can make advances in the field of food sciences to better preserve food and engineer acetic acid-resistant strains that will increase productivity in the biofuel industry. PMID:28432100

Characterisation of monotreme caseins reveals lineage-specific expansion of an ancestral casein locus in mammals.

PubMed

Lefèvre, Christophe M; Sharp, Julie A; Nicholas, Kevin R

2009-01-01

Using a milk-cell cDNA sequencing approach we characterised milk-protein sequences from two monotreme species, platypus (Ornithorhynchus anatinus) and echidna (Tachyglossus aculeatus) and found a full set of caseins and casein variants. The genomic organisation of the platypus casein locus is compared with other mammalian genomes, including the marsupial opossum and several eutherians. Physical linkage of casein genes has been seen in the casein loci of all mammalian genomes examined and we confirm that this is also observed in platypus. However, we show that a recent duplication of beta-casein occurred in the monotreme lineage, as opposed to more ancient duplications of alpha-casein in the eutherian lineage, while marsupials possess only single copies of alpha- and beta-caseins. Despite this variability, the close proximity of the main alpha- and beta-casein genes in an inverted tail-tail orientation and the relative orientation of the more distant kappa-casein genes are similar in all mammalian genome sequences so far available. Overall, the conservation of the genomic organisation of the caseins indicates the early, pre-monotreme development of the fundamental role of caseins during lactation. In contrast, the lineage-specific gene duplications that have occurred within the casein locus of monotremes and eutherians but not marsupials, which may have lost part of the ancestral casein locus, emphasises the independent selection on milk provision strategies to the young, most likely linked to different developmental strategies. The monotremes therefore provide insight into the ancestral drivers for lactation and how these have adapted in different lineages.

Laminin and biomimetic extracellular elasticity enhance functional differentiation in mammary epithelia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Alcaraz, Jordi; Xu, Ren; Mori, Hidetoshi

2008-10-20

In the mammary gland, epithelial cells are embedded in a 'soft' environment and become functionally differentiated in culture when exposed to a laminin-rich extracellular matrix gel. Here, we define the processes by which mammary epithelial cells integrate biochemical and mechanical extracellular cues to maintain their differentiated phenotype. We used single cells cultured on top of gels in conditions permissive for {beta}-casein expression using atomic force microscopy to measure the elasticity of the cells and their underlying substrata. We found that maintenance of {beta}-casein expression required both laminin signalling and a 'soft' extracellular matrix, as is the case in normal tissuesmore » in vivo, and biomimetic intracellular elasticity, as is the case in primary mammary epithelial organoids. Conversely, two hallmarks of breast cancer development, stiffening of the extracellular matrix and loss of laminin signalling, led to the loss of {beta}-casein expression and non-biomimetic intracellular elasticity. Our data indicate that tissue-specific gene expression is controlled by both the tissues unique biochemical milieu and mechanical properties, processes involved in maintenance of tissue integrity and protection against tumorigenesis.« less

Chronic inflammation aggravates metabolic disorders of hepatic fatty acids in high-fat diet-induced obese mice

PubMed Central

Zhao, Lei; Zhong, Shan; Qu, Haiyang; Xie, Yunxia; Cao, Zhennan; Li, Qing; Yang, Ping; Varghese, Zac; Moorhead, John F.; Chen, Yaxi; Ruan, Xiong Z.

2015-01-01

The prevalence of nonalcoholic fatty liver disease (NAFLD) increases with increasing body mass index (BMI). However, approximately 40–50% of obese adults do not develop hepatic steatosis. The level of inflammatory biomarkers is higher in obese subjects with NAFLD compared to BMI-matched subjects without hepatic steatosis. We used a casein injection in high-fat diet (HFD)-fed C57BL/6J mice to induce inflammatory stress. Although mice on a HFD exhibited apparent phenotypes of obesity and hyperlipidemia regardless of exposure to casein injection, only the HFD+Casein mice showed increased hepatic vacuolar degeneration accompanied with elevated inflammatory cytokines in the liver and serum, compared to mice on a normal chow diet. The expression of genes related to hepatic fatty acid synthesis and oxidation were upregulated in the HFD-only mice. The casein injection further increased baseline levels of lipogenic genes and decreased the levels of oxidative genes in HFD-only mice. Inflammatory stress induced both oxidative stress and endoplasmic reticulum stress in HFD-fed mice livers. We conclude that chronic inflammation precedes hepatic steatosis by disrupting the balance between fatty acid synthesis and oxidation in the livers of HFD-fed obese mice. This mechanism may operate in obese individuals with chronic inflammation, thus making them more prone to NAFLD. PMID:25974206

Duplication polymorphisms in exon 4 of κ-casein gene in yak breeds/populations.

PubMed

Pingcuo, S; Gao, J; Jiang, Z R; Jin, S Y; Fu, C Y; Liu, X; Huang, L; Zheng, Y C

2015-08-28

The objective of this study was to compare 12 bp-duplication polymorphisms in exon 4 of the κ-casein gene among 3 breeds/populations of yak (Bos grunniens). Genomic DNA was extracted from yak blood or muscle samples (N = 211) and a partial sequence of exon 4 of κ-casein gene was amplified by polymerase chain reaction. A polyacrylamide gel electrophoresis assay of the products (169 bp) revealed 2 variants. These variants differed in a 12-bp duplication of the nucleotide sequence corresponding to amino acids 147-150 (Glu-Ala-Ser-Pro) or 148-151 (Ala-Ser-Pro-Glu). The genotype frequency and gene frequency of the 2 κ-casein variants differed among the 3 yak breeds/populations. The long form of the κ-casein gene was the predominant allele, and the Jiulong yak showed the highest frequency of the short form variant of the κ-casein gene. In addition, 2 nucleotide differences resulting in amino acid substitutions were also identified in yaks. These results are significant for designing a breeding strategy to improve the genetic makeup of yak herds.

Fish protein hydrolysates affect cholesterol metabolism in rats fed non-cholesterol and high-cholesterol diets.

PubMed

Hosomi, Ryota; Fukunaga, Kenji; Arai, Hirofumi; Kanda, Seiji; Nishiyama, Toshimasa; Yoshida, Munehiro

2012-03-01

Fish consumption is well known to provide health benefits in both experimental animals and human subjects. Numerous studies have demonstrated the beneficial effects of various protein hydrolysates on lipid metabolism. In this context, this study examined the effect of fish protein hydrolysates (FPH) on cholesterol metabolism compared with the effect of casein. FPHs were prepared from Alaska pollock meat using papain as a protease. Male Wistar rats were divided into the following four dietary groups of seven rats each: either casein (20%) or FPH (10%) + casein (10%), with or without 0.5% cholesterol and 0.1% sodium cholate. Serum and liver lipid levels, fecal cholesterol and bile acid excretions, and the hepatic expression of genes encoding proteins involved in cholesterol homeostasis were examined. In rats fed the FPH diets compared with casein diets with or without cholesterol and sodium cholate, the indexes of cholesterol metabolism-namely, serum cholesterol, triglyceride, and low-density lipoprotein-cholesterol levels-were significantly lower, whereas fecal cholesterol and bile acid excretions were higher. Rats fed the FPH diets compared with casein with cholesterol exhibited a lower liver cholesterol level via an increased liver cholesterol 7α-hydroxylase (CYP7A1) expression level. This study demonstrates that the intake of FPH has hypocholesterolemic effects through the enhancement of fecal cholesterol and bile acid excretions and CYP7A1 expression levels. Therefore, fish peptides prepared by papain digestion might provide health benefits by decreasing the cholesterol content in the blood, which would contribute to the prevention of circulatory system diseases such as arteriosclerosis.

Casein Kinase I Isoform Hrr25 Is a Negative Regulator of Haa1 in the Weak Acid Stress Response Pathway in Saccharomyces cerevisiae.

PubMed

Collins, Morgan E; Black, Joshua J; Liu, Zhengchang

2017-07-01

Haa1 is a transcription factor that adapts Saccharomyces cerevisiae cells to weak organic acid stresses by activating the expression of various genes. Many of these genes encode membrane proteins, such as TPO2 and YRO2 How Haa1 is activated by weak acids is not clear. Here, we show that casein kinase I isoform Hrr25 is an important negative regulator of Haa1. Haa1 is known to be multiply phosphorylated. We found that mutations in HRR25 lead to reduced Haa1 phosphorylation and increased expression of Haa1 target genes and that Hrr25 interacts with Haa1. The other three casein kinase I isoforms, Yck1, Yck2, and Yck3, do not seem to play critical roles in Haa1 regulation. Hrr25 has a 200-residue C-terminal region, including a proline- and glutamine-rich domain. Our data suggest that the C-terminal region of Hrr25 is required for normal inhibition of expression of Haa1 target genes TPO2 and YRO2 and is important for cell growth but is not required for cell morphogenesis. We propose that Hrr25 is an important regulator of cellular adaptation to weak acid stress by inhibiting Haa1 through phosphorylation. IMPORTANCE Our study has revealed the casein kinase I protein Hrr25 to be a negative regulator of Haa1, a transcription factor mediating the cellular response to stresses caused by weak acids. Many studies have focused on the target genes of Haa1 and their roles in weak acid stress responses, but little has been reported on the regulatory mechanism of Haa1. Weak acids, such as acetic acid, have long been used for food preservation by slowing down the growth of fungal species, including S. cerevisiae In the biofuel industry, acetic acid in the lignocellulosic hydrolysates limits the production of ethanol, which is undesirable. By understanding how Haa1 is regulated, we can make advances in the field of food sciences to better preserve food and engineer acetic acid-resistant strains that will increase productivity in the biofuel industry. Copyright © 2017 American Society for Microbiology.

Impaired adipogenesis in adipose tissue associated with hepatic lipid deposition induced by chronic inflammation in mice with chew diet.

PubMed

Yang, Shumin; Zhang, Wenlong; Zhen, Qianna; Gao, Rufei; Du, Tingting; Xiao, Xiaoqiu; Wang, Zhihong; Ge, Qian; Hu, Jinbo; Ye, Peng; Zhu, Qibo; Li, Qifu

2015-09-15

Chronic inflammation might be associated with hepatic lipid deposition independent of overnutrition. However, the mechanism is not fully understood. In this study, we investigate if impaired adipogenesis in adipose tissue is associated with hepatic lipid deposition induced by chronic inflammation in mice with chew diet. Casein injection in C57BL/6J mice was given every other day to induce chronic inflammation. All mice were sacrificed after 18weeks of injections. The serum, liver and adipose tissue were collected for analysis. Real-time polymerase chain reaction and western blotting were used to examine the gene and protein expressions of molecules involved in hepatic lipid metabolism and adipose adipogenesis. Casein injection elevated serum levels of insulin, free fatty acid (FFA) and proinflammatory factors. The gene expression of proinflammatory factors of adipose tissue and the liver also increased in the casein group as compared with the control group. Chronic inflammation up-regulated the hepatic expression of fatty acid translocase (CD36) and down-regulated microsomal triacylglycerol transfer protein (MTP), carnitine palmitoyltransferase 1a (CPT1a) and acyl-coenzyme a oxidase 1 (ACOX1). Meanwhile, chronic inflammation not only diminished the size of adipocytes, but also down-regulated the expression of peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer binding proteinα (C/EBPα), both indicating an impaired adipogenesis. Besides disturbed lipid metabolism in the liver per se, impaired adipogenesis in the adipose tissue might also be associated with hepatic lipid deposition induced by chronic inflammation in mice with chew diet. Copyright © 2015 Elsevier Inc. All rights reserved.

White adipose tissue genome wide-expression profiling and adipocyte metabolic functions after soy protein consumption in rats.

PubMed

Frigolet, Maria E; Torres, Nimbe; Uribe-Figueroa, Laura; Rangel, Claudia; Jimenez-Sanchez, Gerardo; Tovar, Armando R

2011-02-01

Obesity is associated with an increase in adipose tissue mass due to an imbalance between high dietary energy intake and low physical activity; however, the type of dietary protein may contribute to its development. The aim of the present work was to study the effect of soy protein versus casein on white adipose tissue genome profiling, and the metabolic functions of adipocytes in rats with diet-induced obesity. The results showed that rats fed a Soy Protein High-Fat (Soy HF) diet gained less weight and had lower serum leptin concentration than rats fed a Casein High-Fat (Cas HF) diet, despite similar energy intake. Histological studies indicated that rats fed the Soy HF diet had significantly smaller adipocytes than those fed the Cas HF diet, and this was associated with a lower triglyceride/DNA content. Fatty acid synthesis in isolated adipocytes was reduced by the amount of fat consumed but not by the type of protein ingested. Expression of genes of fatty acid oxidation increased in adipose tissue of rats fed Soy diets; microarray analysis revealed that Soy protein consumption modified the expression of 90 genes involved in metabolic functions and inflammatory response in adipose tissue. Network analysis showed that the expression of leptin was regulated by the type of dietary protein and it was identified as a central regulator of the expression of lipid metabolism genes in adipose tissue. Thus, soy maintains the size and metabolic functions of adipose tissue through biochemical adaptations, adipokine secretion, and global changes in gene expression. Copyright © 2011 Elsevier Inc. All rights reserved.

Including α s1 casein gene information in genomic evaluations of French dairy goats.

PubMed

Carillier-Jacquin, Céline; Larroque, Hélène; Robert-Granié, Christèle

2016-08-04

Genomic best linear unbiased prediction methods assume that all markers explain the same fraction of the genetic variance and do not account effectively for genes with major effects such as the α s1 casein polymorphism in dairy goats. In this study, we investigated methods to include the available α s1 casein genotype effect in genomic evaluations of French dairy goats. First, the α s1 casein genotype was included as a fixed effect in genomic evaluation models based only on bucks that were genotyped at the α s1 casein locus. Less than 1 % of the females with phenotypes were genotyped at the α s1 casein gene. Thus, to incorporate these female phenotypes in the genomic evaluation, two methods that allowed for this large number of missing α s1 casein genotypes were investigated. Probabilities for each possible α s1 casein genotype were first estimated for each female of unknown genotype based on iterative peeling equations. The second method is based on a multiallelic gene content approach. For each model tested, we used three datasets each divided into a training and a validation set: (1) two-breed population (Alpine + Saanen), (2) Alpine population, and (3) Saanen population. The α s1 casein genotype had a significant effect on milk yield, fat content and protein content. Including an α s1 casein effect in genetic and genomic evaluations based only on male known α s1 casein genotypes improved accuracies (from 6 to 27 %). In genomic evaluations based on all female phenotypes, the gene content approach performed better than the other tested methods but the improvement in accuracy was only slightly better (from 1 to 14 %) than that of a genomic model without the α s1 casein effect. Including the α s1 casein effect in a genomic evaluation model for French dairy goats is possible and useful to improve accuracy. Difficulties in predicting the genotypes for ungenotyped animals limited the improvement in accuracy of the obtained estimated breeding values.

A heterologous hormone response element enhances expression of rat beta-casein promoter-driven chloramphenicol acetyltransferase fusion genes in the mammary gland of transgenic mice.

PubMed

Greenberg, N M; Reding, T V; Duffy, T; Rosen, J M

1991-10-01

Previous studies have demonstrated that the entire rat beta-casein (R beta C) gene and a -524/+490 R beta C fragment-chloramphenicol acetyltransferase (CAT) fusion gene are expressed preferentially in the mammary gland of transgenic mice in a developmentally regulated fashion. However, transgene expression was infrequent, less than 1% of that observed for the endogenous gene, and varied as much as 500-fold, presumably due to the site of chromosomal integration. To determine whether a heterologous hormone-responsive enhancer could be used to increase both the level and frequency of expression in the mammary gland, a fragment derived from the mouse mammary tumor virus long terminal repeat containing four hormone response elements (HREs) was inserted into the R beta C promoter at a site not known to contain transcriptional regulatory elements. Transgenic mice generated which carried HRE-enhanced R beta C-CAT fusion genes expressed CAT activity in the mammary glands of all founder lines examined at levels that were on average 13-fold greater than for lines generated with similar constructs not carrying HREs. In the highest expressing line, the level of HRE-enhanced transgene expression was found to be developmentally regulated, increasing 14-fold in the mammary gland from virgin to day 10 of lactation. In this line, expression was also observed in the thymus and spleen; however, the level of CAT activity was 4-fold lower than in the mammary gland and was not developmentally regulated. In adrenalectomized mice, the administration of dexamethasone stimulated CAT expression in the mammary gland but not in the thymus and spleen. These studies demonstrate that in the context of the R beta C promoter, the HRE functions in the mammary gland to increase both the frequency and level of transgene expression.

Casein gene expression in mouse mammary epithelial cell lines: Dependence upon extracellular matrix and cell type

DOE Office of Scientific and Technical Information (OSTI.GOV)

Medina, D.; Oborn, C.J.; Li, M.L.

1987-09-01

The COMMA-D mammary cell line exhibits mammary-specific functional differentiation under appropriate conditions in cell culture. The cytologically heterogeneous COMMA-D parental line and the clonal lines DB-1, TA-5, and FA-1 derived from the COMMA-D parent were examined for similar properties of functional differentiation. In monolayer cell culture, the cell lines DB-1, TA-5, FA-1, and MA-4 were examined for expression of mammary-specific and epithelial-specific proteins by an indirect immunofluorescence assay. The clonal cell lines were relatively homogeneous in their respective staining properties and seemed to represent three subpopulations found in the heterogeneous parental COMMA-D lines. None of the four clonal lines appearedmore » to represent myoepithelial cells. The cell lines were examined for expression of {beta}-casein mRNA in the presence or absence of prolactin. The inducibility of {beta}-casein in the COMMA-D cell line was further enhanced by a reconstituted basement membrane preparation enriched in laminin, collagen IV, and proteoglycans. These results support the hypothesis that the functional response of inducible mammary cell populations is a result of interaction among hormones, multiple extracellular matrix components, and specific cell types.« less

Immortalized bovine mammary epithelial cells express stem cell markers and differentiate in vitro.

PubMed

Hu, Han; Zheng, Nan; Gao, Haina; Dai, Wenting; Zhang, Yangdong; Li, Songli; Wang, Jiaqi

2016-08-01

The bovine mammary epithelial cell is a secretory cell, and its cell number and secretory activity determine milk production. In this study, we immortalized a bovine mammary epithelial cell line by SV40 large T antigen gene using a retrovirus based on Chinese Holstein primary mammary epithelial cells (CMEC) cultured in vitro. An immortalized bovine mammary epithelial cell line surpassed the 50-passage mark and was designated the CMEC-H. The immortalized mammary epithelial cells grew in close contact with each other and exhibited the typical cobblestone morphology characteristic with obvious boundaries. The telomerase expression of CMEC-H has consistently demonstrated the presence of telomerase activity as an immortalized cell line, but the cell line never induced tumor formation in nude mice. CMEC-H expressed epithelial (cytokeratins CK7, CK8, CK18, and CK19), mesenchymal (vimentin), and stem/progenitor (CD44 and p63) cell markers. The induced expression of milk proteins, αS1 -casein, β-casein, κ-casein, and butyrophilin, indicated that CMEC-H maintained the synthesis function of the mammary epithelial cells. The established immortalized bovine mammary epithelial cell line CMEC-H is capable of self-renewal and differentiation and can serve as a valuable reagent for studying the physiological mechanism of the mammary gland. © 2016 International Federation for Cell Biology.

Milk Lacking α-Casein Leads to Permanent Reduction in Body Size in Mice

PubMed Central

Kolb, Andreas F.; Huber, Reinhard C.; Lillico, Simon G.; Carlisle, Ailsa; Robinson, Claire J.; Neil, Claire; Petrie, Linda; Sorensen, Dorte B.; Olsson, I. Anna S.; Whitelaw, C. Bruce A.

2011-01-01

The major physiological function of milk is the transport of amino acids, carbohydrates, lipids and minerals to mammalian offspring. Caseins, the major milk proteins, are secreted in the form of a micelle consisting of protein and calcium-phosphate. We have analysed the role of the milk protein α-casein by inactivating the corresponding gene in mice. Absence of α-casein protein significantly curtails secretion of other milk proteins and calcium-phosphate, suggesting a role for α-casein in the establishment of casein micelles. In contrast, secretion of albumin, which is not synthesized in the mammary epithelium, into milk is not reduced. The absence of α-casein also significantly inhibits transcription of the other casein genes. α-Casein deficiency severely delays pup growth during lactation and results in a life-long body size reduction compared to control animals, but has only transient effects on physical and behavioural development of the pups. The data support a critical role for α-casein in casein micelle assembly. The results also confirm lactation as a critical window of metabolic programming and suggest milk protein concentration as a decisive factor in determining adult body weight. PMID:21789179

LY294002 inhibits glucocorticoid-induced COX-2 gene expression in cardiomyocytes through a phosphatidylinositol 3 kinase-independent mechanism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun Haipeng; Xu Beibei; Sheveleva, Elena

2008-10-01

Glucocorticoids induce COX-2 expression in rat cardiomyocytes. While investigating whether phosphatidylinositol 3 kinase (PI3K) plays a role in corticosterone (CT)-induced COX-2, we found that LY294002 (LY29) but not wortmannin (WM) attenuates CT from inducing COX-2 gene expression. Expression of a dominant-negative mutant of p85 subunit of PI3K failed to inhibit CT from inducing COX-2 expression. CT did not activate PI3K/AKT signaling pathway whereas LY29 and WM decreased the activity of PI3K. LY303511 (LY30), a structural analogue and a negative control for PI3K inhibitory activity of LY29, also suppressed COX-2 induction. These data suggest PI3K-independent mechanisms in regulating CT-induced COX-2 expression.more » LY29 and LY30 do not inhibit glucocorticoid receptor transactivity. Both compounds have been reported to inhibit Casein Kinase 2 activity and modulate potassium and calcium levels independent of PI3K, while LY29 has been reported to inhibit mammalian Target of Rapamycin (mTOR), and DNA-dependent Protein Kinase (DNA-PK). Inhibitor of Casein Kinase 2 (CK2), mTOR or DNA-PK failed to prevent CT from inducing COX-2 expression. Tetraethylammonium (TEA), a potassium channel blocker, and nimodipine, a calcium channel blocker, both attenuated CT from inducing COX-2 gene expression. CT was found to increase intracellular Ca{sup 2+} concentration, which can be inhibited by LY29, TEA or nimodipine. These data suggest a possible role of calcium instead of PI3K in CT-induced COX-2 expression in cardiomyocytes.« less

Tunable regulation of CREB DNA binding activity couples genotoxic stress response and metabolism

PubMed Central

Kim, Sang Hwa; Trinh, Anthony T.; Larsen, Michele Campaigne; Mastrocola, Adam S.; Jefcoate, Colin R.; Bushel, Pierre R.; Tibbetts, Randal S.

2016-01-01

cAMP response element binding protein (CREB) is a key regulator of glucose metabolism and synaptic plasticity that is canonically regulated through recruitment of transcriptional coactivators. Here we show that phosphorylation of CREB on a conserved cluster of Ser residues (the ATM/CK cluster) by the DNA damage-activated protein kinase ataxia-telangiectasia-mutated (ATM) and casein kinase1 (CK1) and casein kinase2 (CK2) positively and negatively regulates CREB-mediated transcription in a signal dependent manner. In response to genotoxic stress, phosphorylation of the ATM/CK cluster inhibited CREB-mediated gene expression, DNA binding activity and chromatin occupancy proportional to the number of modified Ser residues. Paradoxically, substoichiometric, ATM-independent, phosphorylation of the ATM/CK cluster potentiated bursts in CREB-mediated transcription by promoting recruitment of the CREB coactivator, cAMP-regulated transcriptional coactivators (CRTC2). Livers from mice expressing a non-phosphorylatable CREB allele failed to attenuate gluconeogenic genes in response to DNA damage or fully activate the same genes in response to glucagon. We propose that phosphorylation-dependent regulation of DNA binding activity evolved as a tunable mechanism to control CREB transcriptional output and promote metabolic homeostasis in response to rapidly changing environmental conditions. PMID:27431323

Short communication: Arginase inhibition reduces the synthesis of casein in bovine mammary epithelial cells.

PubMed

Wang, M Z; Ding, L Y; Wang, C; Chen, L M; Loor, J J; Wang, H R

2017-05-01

The main purpose of this work was to determine the effect of arginase inhibition via N ω -hydroxy-nor-l-arginine (nor-NOHA) on casein synthesis in bovine mammary epithelial cells (BMEC). Passage 2 BMEC isolated from dairy cows were seeded to 6-well plates and randomly divided into 4 treatments: (1) control [Dulbecco's modified Eagle medium:Nutrient Mixture F-12 medium (DMEM/F12)]; (2) nor-NOHA (DMEM/F12 + 1 mmol/L nor-NOHA); (3) nor-NOHA + arginine (DMEM/F12 + 1 mmol/L nor-NOHA + 3.2 mmol/L Arg); and (4) nor-NOHA + ornithine (DMEM/F12+ 1 mmol/L nor-NOHA + 1 mmol/L Orn). Then, we determined the activity of enzymes related to Arg metabolism and casein synthesis in BMEC and the proliferation of cells. The addition of nor-NOHA reduced the activity of arginase and ornithine decarboxylase but had no effect on the activity of nitric oxide synthase, and these responses were the same at the gene expression level. In addition, supplementation of nor-NOHA in BMEC reduced cellular proliferation and casein synthesis. Addition of Arg to nor-NOHA resulted in cellular proliferation and casein synthesis similar to that of nor-NOHA alone. In contrast, addition of Orn to the medium with nor-NOHA increased the synthesis of casein and cellular proliferation compared with Nor-NOHA. In conclusion, suppression of the Arg-arginase-Orn pathway reduced casein synthesis and cellular proliferation, which indicated that this pathway is an important regulator of the synthesis of casein in BMEC. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Three-dimensional culture system can induce expression of casein in immortalized bovine mammary epithelial cells.

PubMed

Zhan, Kang; Lin, Miao; Liu, MingMei; Sui, YangNan; Babekir, Haitham Mohammed; Zhao, GuoQi

2017-05-01

Primary bovine mammary epithelial cells (BMECs) are not ideal models for long-term studies of lactation mechanisms because these cells in a monolayer culture system cannot be polarized to simulate the physiological functions in vitro. We investigate the effects of different culture models and karyotypes on casein expression in a three-dimensional (3D) culture system. The immortalized cells' karyotypes were analyzed at passages 10, 20, 30 and 40 to detect the effects of chromosome stability. Western blotting examined that whether or not the immortalized cells at passages 5, 10, 20, 30, 40 and 50 could induce expression of casein in a 3D culture system. The proper polarization of the acinar structures was monitored. BMECs were successfully immortalized. The cell karyotype at passage 30 remained at 60 chromosomes and the average value was 57.1 ± 0.40 after passage 40. The polarized protein's levels were up-regulated in 3D culture compared to 2D culture. Expression of αs1, β and κ-casein could be detectable in a passage range in 3D culture. Expression of αs2-casein was undetectable in all experimental groups. However, all casein expressions were barely detectable in traditional 2D culture system. Therefore, 3D culture system is an important tool for the long-term study of lactation mechanisms in vitro. © 2016 Japanese Society of Animal Science.

Comparative Proteomics Provides Insights into Metabolic Responses in Rat Liver to Isolated Soy and Meat Proteins.

PubMed

Song, Shangxin; Hooiveld, Guido J; Zhang, Wei; Li, Mengjie; Zhao, Fan; Zhu, Jing; Xu, Xinglian; Muller, Michael; Li, Chunbao; Zhou, Guanghong

2016-04-01

It has been reported that isolated dietary soy and meat proteins have distinct effects on physiology and liver gene expression, but the impact on protein expression responses are unknown. Because these may differ from gene expression responses, we investigated dietary protein-induced changes in liver proteome. Rats were fed for 1 week semisynthetic diets that differed only regarding protein source; casein (reference) was fully replaced by isolated soy, chicken, fish, or pork protein. Changes in liver proteome were measured by iTRAQ labeling and LC-ESI-MS/MS. A robust set totaling 1437 unique proteins was identified and subjected to differential protein analysis and biological interpretation. Compared with casein, all other protein sources reduced the abundance of proteins involved in fatty acid metabolism and Pparα signaling pathway. All dietary proteins, except chicken, increased oxidoreductive transformation reactions but reduced energy and essential amino acid metabolic pathways. Only soy protein increased the metabolism of sulfur-containing and nonessential amino acids. Soy and fish proteins increased translation and mRNA processing, whereas only chicken protein increased TCA cycle but reduced immune responses. These findings were partially in line with previously reported transcriptome results. This study further shows the distinct effects of soy and meat proteins on liver metabolism in rats.

Enhancing proliferation and osteogenic differentiation of HMSCs on casein/chitosan multilayer films.

PubMed

Li, Yan; Zheng, Zebin; Cao, Zhinan; Zhuang, Liangting; Xu, Yong; Liu, Xiaozhen; Xu, Yue; Gong, Yihong

2016-05-01

Creating a bioactive surface is important in tissue engineering. Inspired by the natural calcium binding property of casein (CA), multilayer films ((CA/CS)n) with chitosan (CS) as polycation were fabricated to enhance biomineralization, cell adhesion and differentiation. LBL self-assembly technique was used and the assembly process was intensively studied based on changes of UV absorbance, zeta potential and water contact angle. The increasing content of chitosan and casein with bilayers was further confirmed with XPS and TOF-SIMS analysis. To improve the biocompatibility, gelatin was surface grafted. In vitro mineralization test demonstrated that multilayer films had more hydroxyapatite crystal deposition. Human mesenchymal stem cells (HMSCs) were seeded onto these films. According to fluorescein diacetate (FDA) and cell cytoskeleton staining, MTT assay, expression of osteogenic marker genes, ALP activity, and calcium deposition quantification, it was found that these multilayer films significantly promoted HMSCs attachment, proliferation and osteogenic differentiation than TCPS control. Copyright © 2016. Published by Elsevier B.V.

Uterine physiological responses and global gene expression in ovariectomized (ovx) rats treated with soy protein isolate (spi) or 17Beta-estradiol

USDA-ARS?s Scientific Manuscript database

Concerns regarding increased endometrial cancer risk have been raised in women who consume soy products as the result of the estrogenicity of phytochemical components such as the isoflavones genistein and daidzein. Female Sprague-Dawley rats (N = 20/group) were fed AIN-93G diets with casein or SPI a...

Disulphide bonds in casein micelle from milk.

PubMed

Bouguyon, Edwige; Beauvallet, Christian; Huet, Jean-Claude; Chanat, Eric

2006-05-05

Mammary epithelial cells synthesised and secreted caseins, the major milk proteins in most mammals, as large aggregates called micelles into the alveolar lumen they surround. We investigated the implication of the highly conserved cysteine(s) of kappa-casein in disulphide bond formation in casein micelles from several species. Dimers were found in all milks studied, confirming previous observation in ruminants. More importantly, the study of interchain disulphide bridges in mouse and rat casein micelles revealed that any casein possessing a cysteine is engaged in disulphide bond interchange; these species express four or five cysteine-containing caseins, respectively. We found that the main rodent caseins form both homo- and heterodimers. Additionally, disulphide bond formation among milk proteins was specific since the interaction of the caseins with cysteine-containing whey proteins was not observed in native casein micelles.

Inhibitory effect of fluvoxamine on β-casein expression via a serotonin-independent mechanism in human mammary epithelial cells.

PubMed

Chiba, Takeshi; Maeda, Tomoji; Kimura, Soichiro; Morimoto, Yasunori; Sanbe, Atsushi; Ueda, Hideo; Kudo, Kenzo

2015-11-05

Selective serotonin reuptake inhibitors (SSRIs) are widely used as a first-line therapy in postpartum depression. The objective of this study was to determine the mechanism underlying the inhibitory effects of the SSRI, fluvoxamine, on β-casein expression, an indicator of lactation, in MCF-12A human mammary epithelial cells. Expression levels of serotonin (5-hydroxytryptamine; 5-HT) transporter, an SSRI target protein, and tryptophan hydroxylase 1, a rate-limiting enzyme in 5-HT biosynthesis, were increased in MCF-12A cells by prolactin treatment. Treatment with 1 μM fluvoxamine for 72 h significantly decreased protein levels of β-casein and phosphorylated signal transducer and activator transcription 5 (pSTAT5). Extracellular 5-HT levels were significantly increased after exposure to 1 μM fluvoxamine, in comparison with those of untreated and vehicle-treated cells; however, extracellular 5-HT had little effect on the decrease in β-casein expression. Expression of glucose-related protein 78/binding immunoglobulin protein, a regulator of endoplasmic reticulum (ER) stress, was significantly increased after treatment with 1 μM fluvoxamine for 48 h. Exposure to tunicamycin, an inducer of ER stress, also decreased expression of β-casein and pSTAT5 in a manner similar to fluvoxamine. Our results indicate that fluvoxamine suppresses β-casein expression in MCF-12A cells via inhibition of STAT5 phosphorylation caused by induction of ER stress. Further studies are required to confirm the effect of fluvoxamine on the function of mammary epithelial cells. Copyright © 2015 Elsevier B.V. All rights reserved.

Effect of culturing conditions on the expression of key enzymes in the proteolytic system of Lactobacillus bulgaricus *

PubMed Central

Hou, Jun-cai; Liu, Fei; Ren, Da-xi; Han, Wei-wei; Du, Yue-ou

2015-01-01

The proteolytic system of Lactobacillus bulgaricus breaks down milk proteins into peptides and amino acids, which are essential for the growth of the bacteria. The aim of this study was to determine the expressions of seven key genes in the proteolytic system under different culturing conditions (different phases, initial pH values, temperatures, and nitrogen sources) using real-time polymerase chain reaction (RT-PCR). The transcriptions of the seven genes were reduced by 30-fold on average in the stationary phase compared with the exponential growth phase. The transcriptions of the seven genes were reduced by 62.5-, 15.0-, and 59.0-fold in the strains KLDS 08006, KLDS 08007, and KLDS 08012, respectively, indicating that the expressions of the seven genes were significantly different among strains. In addition, the expressions of the seven genes were repressed in the MRS medium containing casein peptone. The effect of peptone supply on PepX transcription was the weakest compared with the other six genes, and the impact on OppD transcription was the strongest. Moreover, the expressions of the seven genes were significantly different among different strains (P<0.05). All these results indicated that the culturing conditions affected the expression of the proteolytic system genes in Lactobacillus bulgaricus at the transcription level. PMID:25845365

Caprine and ovine Greek dairy products: The official German method generates false-positive results due to κ-casein gene polymorphism.

PubMed

Tsartsianidou, V; Triantafillidou, D; Karaiskou, N; Tarantili, P; Triantafillidis, G; Georgakis, E; Triantafyllidis, A

2017-05-01

Caseins are widely used for species identification of dairy products. Isoelectric focusing (IEF) of para-κ-casein peptide is used as the official German method for the differentiation between caprine (isoform A) and ovine (isoform B) dairy products, based on their different isoelectric points. The discrimination between Greek goat and ewe dairy products using IEF has, however, been shown to be problematic because of the existence of the ewe isoform in milk from Greek indigenous dairy goats. This could be due to nucleotide polymorphisms within the goat κ-casein gene of Greek indigenous breeds, which alter the isoelectric point of the para-κ-casein peptide and lead to false positive results. Previous DNA analysis of the goat κ-casein gene has shown high levels of polymorphism; however, no such information is available for Greek indigenous dairy goats. Therefore, 87 indigenous dairy goats were sequenced at exon IV of κ-casein gene. In total, 9 polymorphic sites were detected. Three nonsynonymous point mutations were identified, which change the isoelectric point of the goat para-κ-casein peptide so that it appears identical to that of the ewe peptide. Ten composite genotypes were reconstructed and 6 of them included the problematic point mutations. For the verification of genetic results, IEF was carried out. Both goat and ewe patterns appeared in the problematic genotypes. The frequency of these genotypes could be characterized as moderate (0.23) to high (0.60) within Greek indigenous breeds. However, this is not an issue restricted to Greece, as such genotypes have been detected in various non-Greek goat breeds. In conclusion, IEF based on the official German method is certainly inappropriate for ovine and caprine discrimination concerning Greek dairy goat products, and consequently a new method should be established. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Characteristics and EGFP expression of goat mammary gland epithelial cells.

PubMed

Zheng, Y-M; He, X-Y; Zhang, Y

2010-12-01

The aims of this study were (i) to establish a goat mammary gland epithelial (GMGE) cell line, and (ii) to determine if these GMGE cells could be maintained long-term in culture by continuous subculturing following transfection with a reporter gene, enhanced green fluorescence protein (EGFP). Primary culture of GMGE cells was achieved by outgrowth of migrating cells from the fragments of the mammary gland tissue of a lactating goat. The passage 16 GMGE cells were transfected with EGFP gene using lipofection. The expression of Cell keratins of epithelial cells in GMGE cells was test by immunofluorescence. Βeta-Casein gene mRNA was test for GMGE cells by RT-PCR. The results showed that when grown at low density on a plastic substratum, the GMGE cells formed islands, and when grown to confluency, the cells formed a monolayer and aggregated with the characteristic cobble-stone morphology of epithelial cells. GMGE cells could form dome-like structure which looked like nipple, and the lumen-like structures formed among the cells. Several blister-like structures appeared in the appearance of the cells. The GMGE cells contained different cell types, majority of the cells were short shuttle-like or polygon which were beehive-like. A part of cells were round and flat, a small number of cells were elongated. Some of the GMGE cells contained milk drops. The cell nuclei were round which had 2-4 obvious cores. The expression of Cell keratins demonstrated the property of epithelial cells in GMGE cells by immunofluorescence. The GMGE cells could express transcript encoding a Βeta-Casein protein. EGFP gene was successfully transferred into the GMGE cells, and the transfected cells could be maintained long-term in culture by continuous subculturing. In conclusion, we have established a EGFP gene transfected GMGE (ET-GMGE) cell line and maintained it long-term in culture by continuous subculturing. © 2010 Blackwell Verlag GmbH.

Casein kinase 1α–dependent feedback loop controls autophagy in RAS-driven cancers

PubMed Central

Cheong, Jit Kong; Zhang, Fuquan; Chua, Pei Jou; Bay, Boon Huat; Thorburn, Andrew; Virshup, David M.

2015-01-01

Activating mutations in the RAS oncogene are common in cancer but are difficult to therapeutically target. RAS activation promotes autophagy, a highly regulated catabolic process that metabolically buffers cells in response to diverse stresses. Here we report that casein kinase 1α (CK1α), a ubiquitously expressed serine/threonine kinase, is a key negative regulator of oncogenic RAS–induced autophagy. Depletion or pharmacologic inhibition of CK1α enhanced autophagic flux in oncogenic RAS–driven human fibroblasts and multiple cancer cell lines. FOXO3A, a master longevity mediator that transcriptionally regulates diverse autophagy genes, was a critical target of CK1α, as depletion of CK1α reduced levels of phosphorylated FOXO3A and increased expression of FOXO3A-responsive genes. Oncogenic RAS increased CK1α protein abundance via activation of the PI3K/AKT/mTOR pathway. In turn, elevated levels of CK1α increased phosphorylation of nuclear FOXO3A, thereby inhibiting transactivation of genes critical for RAS-induced autophagy. In both RAS-driven cancer cells and murine xenograft models, pharmacologic CK1α inactivation synergized with lysosomotropic agents to inhibit growth and promote tumor cell death. Together, our results identify a kinase feedback loop that influences RAS-dependent autophagy and suggest that targeting CK1α-regulated autophagy offers a potential therapeutic opportunity to treat oncogenic RAS–driven cancers. PMID:25798617

Serotonin suppresses β-casein expression via PTP1B activation in human mammary epithelial cells.

PubMed

Chiba, Takeshi; Maeda, Tomoji; Sanbe, Atsushi; Kudo, Kenzo

2016-04-22

Serotonin (5-hydroxytriptamine, 5-HT) has an important role in milk volume homeostasis within the mammary gland during lactation. We have previously shown that the expression of β-casein, a differentiation marker in mammary epithelial cells, is suppressed via 5-HT-mediated inhibition of signal transduction and activator of transcription 5 (STAT5) phosphorylation in the human mammary epithelial MCF-12A cell line. In addition, the reduction of β-casein in turn was associated with 5-HT7 receptor expression in the cells. The objective of this study was to determine the mechanisms underlying the 5-HT-mediated suppression of β-casein and STAT5 phosphorylation. The β-casein level and phosphorylated STAT5 (pSTAT5)/STAT5 ratio in the cells co-treated with 5-HT and a protein kinase A (PKA) inhibitor (KT5720) were significantly higher than those of cells treated with 5-HT alone. Exposure to 100 μM db-cAMP for 6 h significantly decreased the protein levels of β-casein and pSTAT5 and the pSTAT5/STAT5 ratio, and significantly increased PTP1B protein levels. In the cells co-treated with 5-HT and an extracellular signal-regulated kinase1/2 (ERK) inhibitor (FR180294) or Akt inhibitor (124005), the β-casein level and pSTAT5/STAT5 ratio were equal to those of cells treated with 5-HT alone. Treatment with 5-HT significantly induced PTP1B protein levels, whereas its increase was inhibited by KT5720. In addition, the PTP1B inhibitor sc-222227 increased the expression levels of β-casein and the pSTAT5/STAT5 ratio. Our observations indicate that PTP1B directly regulates STAT5 phosphorylation and that its activation via the cAMP/PKA pathway downstream of the 5-HT7 receptor is involved in the suppression of β-casein expression in MCF-12A cells. Copyright © 2016 Elsevier Inc. All rights reserved.

Cloning, expression, and mapping of allergenic determinants of alphaS1-casein, a major cow's milk allergen.

PubMed

Schulmeister, Ulrike; Hochwallner, Heidrun; Swoboda, Ines; Focke-Tejkl, Margarete; Geller, Beate; Nystrand, Mats; Härlin, Annika; Thalhamer, Josef; Scheiblhofer, Sandra; Keller, Walter; Niggemann, Bodo; Quirce, Santiago; Ebner, Christoph; Mari, Adriano; Pauli, Gabrielle; Herz, Udo; Valenta, Rudolf; Spitzauer, Susanne

2009-06-01

Milk is one of the first components introduced into human diet. It also represents one of the first allergen sources, which induces IgE-mediated allergies in childhood ranging from gastrointestinal, skin, and respiratory manifestations to severe life-threatening manifestations, such as anaphylaxis. Here we isolated a cDNA coding for a major cow's milk allergen, alphaS1-casein, from a bovine mammary gland cDNA library with allergic patients' IgE Abs. Recombinant alphaS1-casein was expressed in Escherichia coli, purified, and characterized by circular dichroism as a folded protein. IgE epitopes of alphaS1-casein were determined with recombinant fragments and synthetic peptides spanning the alphaS1-casein sequence using microarrayed components and sera from 66 cow's milk-sensitized patients. The allergenic activity of ralphaS1-casein and the alphaS1-casein-derived peptides was determined using rat basophil leukemia cells transfected with human FcepsilonRI, which had been loaded with the patients' serum IgE. Our results demonstrate that ralphaS1-casein as well as alphaS1-casein-derived peptides exhibit IgE reactivity, but mainly the intact ralphaS1-casein induced strong basophil degranulation. These results suggest that primarily intact alphaS1-casein or larger IgE-reactive portions thereof are responsible for IgE-mediated symptoms of food allergy. Recombinant alphaS1-casein as well as alphaS1-casein-derived peptides may be used in clinical studies to further explore pathomechanisms of food allergy as well as for the development of new diagnostic and therapeutic strategies for milk allergy.

Feeding soy protein isolate and treatment with estradiol have different effects on mammary gland morphology and gene expression in weanling male and female rats

USDA-ARS?s Scientific Manuscript database

Isoflavones are phytochemical components of soy diets that bind weakly to estrogen receptors (ERs). To study potential estrogen-like actions of soy in the mammary gland during early development, we fed weanling male and female Sprague-Dawley rats a semi-purified diet with casein as the sole protein ...

Estrogen regulation of uterine genes in vivo detected by complementary DNA array.

PubMed

Andrade, P M; Silva, I D C G; Borra, R C; de Lima, G R; Baracat, E C

2002-05-01

In the present study, our aim was to identify differentially expressed genes involved in estrogen actions at the endometrium level in rats. Thirty adult rats were ovariectomized four days prior to drug administration for 48 days. Rats were divided in 2 groups: I, control and II, conjugated equine estrogens (CCE). Total RNA was isolated from uterus, and differential expression was analyzed by array technology and RT-PCR. A total of 32 candidate genes were shown to be upregulated or downregulated in groups I or II. Among them, differential expression was already confirmed by RT-PCR for IGFBP5, S12, c-kit, and VEGF, genes whose expression was up regulated during CCE therapy, and casein kinase II and serine kinase expression was the same level in both groups. We have demonstrated that cDNA array represents a powerful approach to identify key molecules in the estrogens therapy. A number of the candidates reported here should provide new markers that may contribute to the detection of target estrogen receptor. This information may also aid the development of new approaches to therapeutic intervention.

Relative quantification of beta-casein expression in primary goat mammary epithelial cell lines.

PubMed

Ogorevc, J; Dovč, P

2015-04-15

Primary mammary epithelial cell cultures were established from mammary tissue of lactating and non-lactating goats to assess the expression of beta-casein (CSN2) in vitro. Primary cell cultures were established by enzymatic digestion of mammary tissue and characterized using antibodies against cytokeratin 14, cytokeratin 18, and vimentin. The established primary cell lines in the second passage were grown in basal medium on plastic and in hormone-supplemented (lactogenic) medium on plastic and on an extracellular matrix-covered surface, respectively. CSN2 gene expression was evaluated using quantitative reverse transcription PCR. The presence of CSN2 transcripts was detected in all samples, including cells originating from non-lactating goat, grown in basal medium. The presence of CSN2 protein was confirmed using immunofluorescence. Response to the hormonal treatment and cell morphology differed between the cell lines and treatments. In 2 cell lines supplemented with lactogenic hormones in the medium, CSN2 expression was increased, while CSN2 levels in one of the cell lines remained constant, regardless of the treatment. Addition of extracellular matrix showed positive effects on CSN2 transcription activity in 1 of the cell lines, while in the other 2 showed no statistically significant effects. CSN2 expression appeared to depend on subtle differences in physiological state of the starting tissue material, growth conditions, cell types present in the culture, and methods used for cell culture establishment. Further studies are necessary to identify factors that determine hormone-responsiveness and transcriptional activity of milk protein genes in goat primary mammary cell cultures.

Refeeding with a high-protein diet after a 48 h fast causes acute hepatocellular injury in mice.

PubMed

Oarada, Motoko; Tsuzuki, Tsuyoshi; Nikawa, Takeshi; Kohno, Shohei; Hirasaka, Katsuya; Gonoi, Tohru

2012-05-01

Elucidating the effects of refeeding a high-protein diet after fasting on disease development is of interest in relation to excessive protein ingestion and irregular eating habits in developed countries. The objective of the present study was to address the hepatic effects of refeeding a high-protein diet after fasting. Mice were fasted for 48 h and then refed with a test diet containing 3, 15, 35, 40, 45 or 50 % casein. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities and liver immediate-early gene expression levels were sequentially measured for the first 24 h after initiation of refeeding. Refeeding with a 50 % casein diet after 48 h of fasting led to a rapid (within 2-3 h) and abnormal elevation in serum ALT (P = 0·006) and AST (P = 0·001) activities and a marked increase in liver Finkel-Biskis-Jinkins (FBJ) osteosarcoma oncogene (P = 0·007) and nuclear receptor subfamily 4, group A, member 1 (P = 0·002) mRNA levels. In contrast, refeeding of the 3, 15 or 35 % casein diets produced no substantial increases in serum ALT and AST activities in mice. Refeeding of 40, 45 or 50 % casein increased serum ALT and AST activities in proportion to this dietary casein content. In mice refed the 3, 15 or 35, but not 50 %, casein diets, liver heat shock protein 72 transcript levels greatly increased. We conclude from these data that the consumption of a high-protein diet after fasting causes acute hepatocellular injury in healthy animals, and propose that careful attention should be paid to the use of such diets.

Nucleotide sequence determination of guinea-pig casein B mRNA reveals homology with bovine and rat alpha s1 caseins and conservation of the non-coding regions of the mRNA.

PubMed Central

Hall, L; Laird, J E; Craig, R K

1984-01-01

Nucleotide sequence analysis of cloned guinea-pig casein B cDNA sequences has identified two casein B variants related to the bovine and rat alpha s1 caseins. Amino acid homology was largely confined to the known bovine or predicted rat phosphorylation sites and within the 'signal' precursor sequence. Comparison of the deduced nucleotide sequence of the guinea-pig and rat alpha s1 casein mRNA species showed greater sequence conservation in the non-coding than in the coding regions, suggesting a functional and possibly regulatory role for the non-coding regions of casein mRNA. The results provide insight into the evolution of the casein genes, and raise questions as to the role of conserved nucleotide sequences within the non-coding regions of mRNA species. Images Fig. 1. PMID:6548375

A 3,387 bp 5'-flanking sequence of the goat alpha-S1-casein gene provides correct tissue-specific expression of human granulocyte colony-stimulating factor (hG-CSF) in the mammary gland of transgenic mice.

PubMed

Serova, Irina A; Dvoryanchikov, Gennady A; Andreeva, Ludmila E; Burkov, Ivan A; Dias, Luciene P B; Battulin, Nariman R; Smirnov, Alexander V; Serov, Oleg L

2012-06-01

A new expression vector containing the 1,944 bp 5'-flanking regulatory region together with exon 1 and intron 1 of the goat alpha-S1-casein gene (CSN1S1), the full-sized human granulocyte colony-stimulating factor gene (hGCSF) and the 3'-flanking sequence of the bovine CSN1S1, was created. The vector DNA was used for generation of four mouse transgenic lines. The transgene was integrated into chromosomes 8 and 12 of two founders as 2 and 5 copies, respectively. Tissue-specific secretion of hG-CSF into the milk of transgenic mice was in the range of 19-40 μg/ml. RT-PCR analysis of various tissues of the transgenic mice demonstrated that expression of hGCSF was detected in only the mammary gland in the progeny of all founders. Moreover, cells were shown to be positive for hG-CSF by immunofluorescent analysis in the mammary glands but not in any other tissues. There were no signs of mosaic expression in the mammary gland. Trace amounts of hG-CSF were detected in the serum of females of two transgenic lines during lactation only. However, no transgenic mice showed any changes in hematopoiesis based on the number of granulocytes in blood. Immunoblotting of hG-CSF in the milk of transgenic mice revealed two forms, presumably the glycosylated and non-glycosylated forms. The hematopoietic activity of hG-CSF in the milk of transgenic females is comparable to that of recombinant G-CSF. In general, the data obtained in this study show that the new expression vector is able to provide correct tissue-specific expression of hG-CSF with high biological activity in transgenic mice.

Feeding soy protein isolate (SPI) does not result in an estrogenic gene expression profile in the mammary of ovariectomized (OVX) female rats

USDA-ARS?s Scientific Manuscript database

Concerns of increased breast cancer risk in women consuming soy exist because of the perceived estrogenicity of soy isoflavones. Female Sprague-Dawley rats (N equals 20/group) were fed AIN-93G diets with casein or SPI as the protein from PND30. On PND50 rats were OVX and 10/group infused s.c. with 5...

Diet and sex modify exercise and cardiac adaptation in the mouse

PubMed Central

Chen, Hao; Luczak, Elizabeth; McKee, Laurel A.; Regan, Jessica; Watson, Peter A.; Stauffer, Brian L.; Khalpey, Zain I; Mckinsey, Timothy A.; Horn, Todd; LaFleur, Bonnie; Leinwand, Leslie A.

2014-01-01

The heart adapts to exercise stimuli in a sex-dimorphic manner when mice are fed the traditional soy-based chow. Females undergo more voluntary exercise (4 wk) than males and exhibit more cardiac hypertrophy per kilometer run (18, 32). We have found that diet plays a critical role in cage wheel exercise and cardiac adaptation to the exercise stimulus in this sex dimorphism. Specifically, feeding male mice a casein-based, soy-free diet increases daily running distance over soy-fed counterparts to equal that of females. Moreover, casein-fed males have a greater capacity to increase their cardiac mass in response to exercise compared with soy-fed males. To further explore the biochemical mechanisms for these differences, we performed a candidate-based RT-PCR screen on genes previously implicated in diet- or exercise-based cardiac hypertrophy. Of the genes screened, many exhibit significant exercise, diet, or sex effects but only transforming growth factor-β1 shows a significant three-way interaction with no genes showing a two-way interaction. Finally, we show that the expression and activity of adenosine monophosphate-activated kinase-α2 and acetyl-CoA carboxylase is dependent on exercise, diet, and sex. PMID:25398983

Diet and sex modify exercise and cardiac adaptation in the mouse.

PubMed

Konhilas, John P; Chen, Hao; Luczak, Elizabeth; McKee, Laurel A; Regan, Jessica; Watson, Peter A; Stauffer, Brian L; Khalpey, Zain I; Mckinsey, Timothy A; Horn, Todd; LaFleur, Bonnie; Leinwand, Leslie A

2015-01-15

The heart adapts to exercise stimuli in a sex-dimorphic manner when mice are fed the traditional soy-based chow. Females undergo more voluntary exercise (4 wk) than males and exhibit more cardiac hypertrophy per kilometer run (18, 32). We have found that diet plays a critical role in cage wheel exercise and cardiac adaptation to the exercise stimulus in this sex dimorphism. Specifically, feeding male mice a casein-based, soy-free diet increases daily running distance over soy-fed counterparts to equal that of females. Moreover, casein-fed males have a greater capacity to increase their cardiac mass in response to exercise compared with soy-fed males. To further explore the biochemical mechanisms for these differences, we performed a candidate-based RT-PCR screen on genes previously implicated in diet- or exercise-based cardiac hypertrophy. Of the genes screened, many exhibit significant exercise, diet, or sex effects but only transforming growth factor-β1 shows a significant three-way interaction with no genes showing a two-way interaction. Finally, we show that the expression and activity of adenosine monophosphate-activated kinase-α2 and acetyl-CoA carboxylase is dependent on exercise, diet, and sex.

Hypolipidemic effect of dietary pea proteins: Impact on genes regulating hepatic lipid metabolism.

PubMed

Rigamonti, Elena; Parolini, Cinzia; Marchesi, Marta; Diani, Erika; Brambilla, Stefano; Sirtori, Cesare R; Chiesa, Giulia

2010-05-01

Controversial data on the lipid-lowering effect of dietary pea proteins have been provided and the mechanisms behind this effect are not completely understood. The aim of the study was to evaluate a possible hypolipidemic activity of a pea protein isolate and to determine whether pea proteins could affect the hepatic lipid metabolism through regulation of genes involved in cholesterol and fatty acid homeostasis. Rats were fed Nath's hypercholesterolemic diets for 28 days, the protein sources being casein or a pea protein isolate from Pisum sativum. After 14 and 28 days of dietary treatment, rats fed pea proteins had markedly lower plasma cholesterol and triglyceride levels than rats fed casein (p<0.05). Pea protein-fed rats displayed higher hepatic mRNA levels of LDL receptor versus those fed casein (p<0.05). Hepatic mRNA concentration of genes involved in fatty acids synthesis, such as fatty acid synthase and stearoyl-CoA desaturase, was lower in pea protein-fed rats than in rats fed casein (p<0.05). In conclusion, the present study demonstrates a marked cholesterol and triglyceride-lowering activity of pea proteins in rats. Moreover, pea proteins appear to affect cellular lipid homeostasis by upregulating genes involved in hepatic cholesterol uptake and by downregulating fatty acid synthesis genes.

Yeast casein kinase 2 governs morphology, biofilm formation, cell wall integrity, and host cell damage of Candida albicans.

PubMed

Jung, Sook-In; Rodriguez, Natalie; Irrizary, Jihyun; Liboro, Karl; Bogarin, Thania; Macias, Marlene; Eivers, Edward; Porter, Edith; Filler, Scott G; Park, Hyunsook

2017-01-01

The regulatory networks governing morphogenesis of a pleomorphic fungus, Candida albicans are extremely complex and remain to be completely elucidated. This study investigated the function of C. albicans yeast casein kinase 2 (CaYck2p). The yck2Δ/yck2Δ strain displayed constitutive pseudohyphae in both yeast and hyphal growth conditions, and formed enhanced biofilm under non-biofilm inducing condition. This finding was further supported by gene expression analysis of the yck2Δ/yck2Δ strain which showed significant upregulation of UME6, a key transcriptional regulator of hyphal transition and biofilm formation, and cell wall protein genes ALS3, HWP1, and SUN41, all of which are associated with morphogenesis and biofilm architecture. The yck2Δ/yck2Δ strain was hypersensitive to cell wall damaging agents and had increased compensatory chitin deposition in the cell wall accompanied by an upregulation of the expression of the chitin synthase genes, CHS2, CHS3, and CHS8. Absence of CaYck2p also affected fungal-host interaction; the yck2Δ/yck2Δ strain had significantly reduced ability to damage host cells. However, the yck2Δ/yck2Δ strain had wild-type susceptibility to cyclosporine and FK506, suggesting that CaYck2p functions independently from the Ca+/calcineurin pathway. Thus, in C. albicans, Yck2p is a multifunctional kinase that governs morphogenesis, biofilm formation, cell wall integrity, and host cell interactions.

Nucleotide sequences of bovine alpha S1- and kappa-casein cDNAs.

PubMed Central

Stewart, A F; Willis, I M; Mackinlay, A G

1984-01-01

The nucleotide sequences corresponding to bovine alpha S1- and kappa-casein mRNAs are presented. An unusual alpha S1-casein cDNA has been characterised whose 5' end commences upstream from its putative TATA box. The alpha S1-casein mRNA is compared to rat alpha-casein mRNA and two components of divergence are identified. Firstly, the two sequences have diverged at a high point mutation rate and the rate of amino acid replacement by this mechanism is at least as great as the rate of divergence of any other part of the mRNAs. Secondly, the protein coding sequence has been subjected to several insertion/deletion events, one of which may be an example of exon shuffling . The kappa-casein mRNA sequence verifies the proposition that it has arisen from a different ancestral gene to the other caseins. Images PMID:6328443

Soy protein supports cardiovascular health by downregulating hydroxymethylglutaryl-coenzyme A reductase and sterol regulatory element-binding protein-2 and increasing antioxidant enzyme activity in rats with dextran sodium sulfate-induced mild systemic inflammation.

PubMed

Marsh, Tanya G; Straub, Rachel K; Villalobos, Fatima; Hong, Mee Young

2011-12-01

Animal and human studies have indicated that the presence of soy in the diet improves cardiovascular health. Inflammation plays a pivotal role in the progression of cardiovascular disease (CVD). However, little is known about how dextran sodium sulfate (DSS)-induced systemic inflammation impacts overall heart health and, correspondingly, how soy protein modulates risk of CVD development in DSS-induced systemic inflammation. We hypothesized that soy protein-fed rats would have a lower risk of CVD by beneficial alteration of gene expression involving lipid metabolism and antioxidant capacity in DSS-induced systemic inflammation. Forty Sprague-Dawley rats were divided into 4 groups: casein, casein + DSS, soy protein, and soy protein + DSS. After 26 days, inflammation was induced in one group from each diet by incorporating 3% DSS in drinking water for 48 hours. Soy protein-fed rats had lower final body weights (P = .010), epididymal fat weights (P = .049), total cholesterol (P < .001), and low-density lipoprotein cholesterol (P < .001). In regard to gene expression, soy protein-fed rats had lower sterol regulatory element-binding protein-2 (P = .032) and hydroxymethylglutaryl-coenzyme A reductase (P = .028) levels and higher low-density lipoprotein receptor levels (P = .036). Antioxidant enzyme activity of superoxide dismutase and catalase was higher among the soy protein groups (P = .037 and P = .002, respectively). These results suggest that soy protein positively influences cardiovascular health by regulating serum lipids through modified expression of sterol regulatory element-binding protein-2 and its downstream genes (ie, hydroxymethylglutaryl-coenzyme A reductase and low-density lipoprotein receptor) and by promoting the antioxidant enzyme activity of superoxide dismutase and catalase. Copyright © 2011 Elsevier Inc. All rights reserved.

Helicobacter pylori induces cell migration and invasion through casein kinase 2 in gastric epithelial cells.

PubMed

Lee, Yeo Song; Lee, Do Yeon; Yu, Da Yeon; Kim, Shin; Lee, Yong Chan

2014-12-01

Chronic infection with Helicobacter pylori (H. pylori) is causally linked with gastric carcinogenesis. Virulent H. pylori strains deliver bacterial CagA into gastric epithelial cells. Induction of high motility and an elongated phenotype is considered to be CagA-dependent process. Casein kinase 2 plays a critical role in carcinogenesis through signaling pathways related to the epithelial mesenchymal transition. This study was aimed to investigate the effect of H. pylori infection on the casein kinase 2-mediated migration and invasion in gastric epithelial cells. AGS or MKN28 cells as human gastric epithelial cells and H. pylori strains Hp60190 (ATCC 49503, CagA(+)) and Hp8822 (CagA(-)) were used. Cells were infected with H. pylori at multiplicity of infection of 100 : 1 for various times. We measured in vitro kinase assay to examine casein kinase 2 activity and performed immunofluorescent staining to observe E-cadherin complex. We also examined β-catenin transactivation through promoter assay and MMP7 expression by real-time PCR and ELISA. H. pylori upregulates casein kinase 2 activity and inhibition of casein kinase 2 in H. pylori-infected cells profoundly suppressed cell invasiveness and motility. We confirmed that casein kinase 2 mediates membranous α-catenin depletion through dissociation of the α-/β-catenin complex in H. pylori-infected cells. We also found that H. pylori induces β-catenin nuclear translocation and increases MMP7 expressions mediated through casein kinase 2. We show for the first time that CagA(+) H. pylori upregulates cellular invasiveness and motility through casein kinase 2. The demonstration of a mechanistic interplay between H. pylori and casein kinase 2 provides important insights into the role of CagA(+) H. pylori in the gastric cancer invasion and metastasis. © 2014 John Wiley & Sons Ltd.

κ-Casein terminates casein micelle build-up by its "soft" secondary structure.

PubMed

Nagy, Krisztina; Váró, György; Szalontai, Balázs

2012-11-01

In our previous paper (Nagy et al. in J Biol Chem 285:38811-38817, 2010) by using a multilayered model system, we showed that, from α-casein, aggregates (similar to natural casein micelles) can be built up step by step if Ca-phosphate nanocluster incorporation is ensured between the protein adsorption steps. It remained, however, an open question whether the growth of the aggregates can be terminated, similarly to in nature with casein micelles. Here, we show that, in the presence of Ca-phosphate nanoclusters, upon adsorbing onto earlier α-casein surfaces, the secondary structure of α-casein remains practically unaffected, but κ-casein exhibits considerable changes in its secondary structure as manifested by a shift toward having more β-structures. In the absence of Ca-phosphate, only κ-casein can still adsorb onto the underlying casein surface; this κ-casein also expresses considerable shift toward β-structures. In addition, this κ-casein cover terminates casein aggregation; no further adsorption of either α- or κ-casein can be achieved. These results, while obtained on a model system, may show that the Ca-insensitive κ-casein can, indeed, be the outer layer of the casein micelles, not only because of its "hairy" extrusion into the water phase, but because of its "softer" secondary structure, which can "occlude" the interacting motifs serving casein aggregation. We think that the revealed nature of the molecular interactions, and the growth mechanism found here, might be useful to understand the aggregation process of casein micelles also in vivo.

Transport of beta-casein-derived peptides by the oligopeptide transport system is a crucial step in the proteolytic pathway of Lactococcus lactis.

PubMed

Kunji, E R; Hagting, A; De Vries, C J; Juillard, V; Haandrikman, A J; Poolman, B; Konings, W N

1995-01-27

In the proteolytic pathway of Lactococcus lactis, milk proteins (caseins) are hydrolyzed extracellularly to oligopeptides by the proteinase (PrtP). The fate of these peptides, i.e. extracellular hydrolysis followed by amino acid uptake or transport followed by intracellular hydrolysis, has been addressed. Mutants have been constructed that lack a functional di-tripeptide transport system (DtpT) and/or oligopeptide transport system (Opp) but do express the P1-type proteinase (specific for hydrolysis of beta- and to a lesser extent kappa-casein). The wild type strain and the DtpT- mutant accumulate all beta-casein-derived amino acids in the presence of beta-casein as protein substrate and glucose as a source of metabolic energy. The amino acids are not accumulated significantly inside the cells by the Opp- and DtpT- Opp- mutants. When cells are incubated with a mixture of amino acids mimicking the composition of beta-casein, the amino acids are taken up to the same extent in all four strains. Analysis of the extracellular peptide fraction, formed by the action of PrtP on beta-casein, indicates that distinct peptides disappear only when the cells express an active Opp system. These and other experiments indicate that (i) oligopeptide transport is essential for the accumulation of all beta-casein-derived amino acids, (ii) the activity of the Opp system is sufficiently high to support high growth rates on beta-casein provided leucine and histidine are present as free amino acids, and (iii) extracellular peptidase activity is not present in L. lactis.

Variability of hydrolysis of β-, αs1-, and αs2-caseins by 10 strains of Streptococcus thermophilus and resulting bioactive peptides.

PubMed

Miclo, Laurent; Roux, Emeline; Genay, Magali; Brusseaux, Emilie; Poirson, Chantal; Jameh, Nawara; Perrin, Clarisse; Dary, Annie

2012-01-18

Milk proteins contain numerous potential bioactive peptides, which may be released by digestive proteases or by the proteolytic system of lactic acid bacteria during food processing. The capacity of Streptococcus thermophilus to generate peptides, especially bioactive peptides, from bovine caseins was investigated. Strains expressing various levels of the cell envelope proteinase, PrtS, were incubated with α(s1)-, α(s2)-, or β-casein. Analysis of the supernatants by LC-ESI-MS/MS showed that the β-casein was preferentially hydrolyzed, followed by α(s2)-casein and then α(s1)-casein. Numbers and types of peptides released were strain-dependent. Hydrolysis appeared to be linked with the accessibility of different casein regions by protease. Analysis of bonds hydrolyzed in the region 1-23 of α(s1)-casein suggests that PrtS is at least in part responsible for the peptide production. Finally, among the generated peptides, 13 peptides from β-casein, 5 from α(s2)-casein, and 2 from α(s1)-casein have been reported as bioactive, 15 of them being angiotensin-converting enzyme inhibitors.

TLR and NLRP3 inflammasome expression deregulation in macrophages of adult rats subjected to neonatal malnutrition and infected with methicillin-resistant Staphylococcus aureus.

PubMed

Gomes de Morais, Natália; Barreto da Costa, Thacianna; Bezerra de Lira, Joana Maria; da Cunha Gonçalves de Albuquerque, Suênia; Alves Pereira, Valéria Rêgo; de Paiva Cavalcanti, Milena; Machado Barbosa de Castro, Célia Maria

2017-01-01

Nutritional aggression in critical periods may lead to epigenetic changes that affect gene expression. The aim of this study was to assess the effect of neonatal malnutrition on the expression of toll-like receptor (TLR)-2, TLR-4, and NLRP3 receptors, caspase-1 enzyme, and interleukin (IL)-1 β production in macrophages infected with methicillin-resistant (MRSA) and methicillin-sensitive (MSSA) Staphylococcus aureus. Wistar rats (N = 24) were divided in two distinct groups: nourished (17% casein) and malnourished (8% casein). Four systems were established after the isolation of mononuclear cells: negative control, positive control, MRSA, and MSSA. The plates were incubated at 37°C for 24 h in humidified atmosphere and 5% carbon dioxide. Tests were performed after this period to analyze the expression of standard recognition receptors, caspase-1 enzyme, and the production of IL-1 β. Student's t test and analysis of variance were used in the statistical analysis; P < 0.05 was statistically significant. Malnutrition reduced animal growth and the expression of TLR-2, TLR-4, and NLRP3 receptors, the caspase-1 enzyme, and the IL-1 β levels in macrophages infected with lipopolysaccharides in the present study. However, the interaction between the S. aureus and the macrophages promoted greater gene expression of receptors and enzymes. The neonatal malnutrition model compromised the expression of standard recognition receptors, of the caspase-1 enzyme as well as the production of IL-1 β. However, the S. aureus and neonatal malnutrition combination led to intense transcription of such innate immunity components. Therefore, the deregulation in the expression of TLR and NLRP3 receptors and of the caspase-1 enzyme may induce extensive tissue injury and favor the permanence and spread of these bacteria, especially those that are methicillin resistant. Copyright © 2016 Elsevier Inc. All rights reserved.

Characterization and Comparative Analysis of the Milk Transcriptome in Two Dairy Sheep Breeds using RNA Sequencing.

PubMed

Suárez-Vega, Aroa; Gutiérrez-Gil, Beatriz; Klopp, Christophe; Robert-Granie, Christèle; Tosser-Klopp, Gwenola; Arranz, Juan José

2015-12-18

This study presents a dynamic characterization of the sheep milk transcriptome aiming at achieving a better understanding of the sheep lactating mammary gland. Transcriptome sequencing (RNA-seq) was performed on total RNA extracted from milk somatic cells from ewes on days 10, 50, 120 and 150 after lambing. The experiment was performed in Spanish Churra and Assaf breeds, which differ in their milk production traits. Nearly 67% of the annotated genes in the reference genome (Oar_v3.1) were expressed in ovine milk somatic cells. For the two breeds and across the four lactation stages studied, the most highly expressed genes encoded caseins and whey proteins. We detected 573 differentially expressed genes (DEGs) across lactation points, with the largest differences being found, between day 10 and day 150. Upregulated GO terms at late lactation stages were linked mainly to developmental processes linked to extracellular matrix remodeling. A total of 256 annotated DEGs were detected in the Assaf and Churra comparison. Some genes selectively upregulated in the Churra breed grouped under the endopeptidase and channel activity GO terms. These genes could be related to the higher cheese yield of this breed. Overall, this study provides the first integrated overview on sheep milk gene expression.

Characterization and Comparative Analysis of the Milk Transcriptome in Two Dairy Sheep Breeds using RNA Sequencing

PubMed Central

Suárez-Vega, Aroa; Gutiérrez-Gil, Beatriz; Klopp, Christophe; Robert-Granie, Christèle; Tosser-Klopp, Gwenola; Arranz, Juan José

2015-01-01

This study presents a dynamic characterization of the sheep milk transcriptome aiming at achieving a better understanding of the sheep lactating mammary gland. Transcriptome sequencing (RNA-seq) was performed on total RNA extracted from milk somatic cells from ewes on days 10, 50, 120 and 150 after lambing. The experiment was performed in Spanish Churra and Assaf breeds, which differ in their milk production traits. Nearly 67% of the annotated genes in the reference genome (Oar_v3.1) were expressed in ovine milk somatic cells. For the two breeds and across the four lactation stages studied, the most highly expressed genes encoded caseins and whey proteins. We detected 573 differentially expressed genes (DEGs) across lactation points, with the largest differences being found, between day 10 and day 150. Upregulated GO terms at late lactation stages were linked mainly to developmental processes linked to extracellular matrix remodeling. A total of 256 annotated DEGs were detected in the Assaf and Churra comparison. Some genes selectively upregulated in the Churra breed grouped under the endopeptidase and channel activity GO terms. These genes could be related to the higher cheese yield of this breed. Overall, this study provides the first integrated overview on sheep milk gene expression. PMID:26677795

Separation and partial characterization of guinea-pig caseins.

PubMed Central

Craig, R K; McIlreavy, D; Hall, R L

1978-01-01

1. Guinea-pig caseins A, B and C were purified free of each other by a combination of ion-exchange chromatography and gel filtration. 2. Determination of the amino acid composition showed all three caseins to contain a high proportion of proline and glutamic acid, but no cysteine. This apart, the amino acid composition of the three caseins was markedly different, though calculated divergence values suggest that some homology may exist between caseins A and B. Molecular-weight estimates based on amino acid composition were in good agreement with those based on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 3. N-Terminal analysis showed lysine, methionine and lysine to be the N-terminal residues of caseins A, B and C respectively. 4. Two-dimensional separation of tryptic digests revealed a distinctive pattern for each casein. 5. All caseins were shown to be phosphoproteins. The casein C preparation also contained significant amounts of sialic acid, neutral and amino sugars. 6. The results suggest that each casein represents a separate gene product, and that the low-molecular-weight proteins are not the result of a post-translational cleavage of the largest. All were distinctly different from the whey protein alpha-lactalbumin. Images Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:697741

The study of zinc ions binding to casein.

PubMed

Pomastowski, P; Sprynskyy, M; Buszewski, B

2014-08-01

The presented research was focused on physicochemical study of casein properties and the kinetics of zinc ions binding to the protein. Moreover, a fast and simple method of casein extraction from cow's milk has been proposed. Casein isoforms, zeta potential (ζ) and particle size of the separated caseins were characterized with the use of capillary electrophoresis, zeta potential analysis and field flow fractionation (FFF) technique, respectively. The kinetics of the metal-binding process was investigated in batch adsorption experiments. Intraparticle diffusion model, first-order and zero-order kinetic models were applied to test the kinetic experimental data. Analysis of changes in infrared bands registered for casein before and after zinc binding was also performed. The obtained results showed that the kinetic process of zinc binding to casein is not homogeneous but is expressed with an initial rapid stage with about 70% of zinc ions immobilized by casein and with a much slower second step. Maximum amount of bound zinc in the experimental conditions was 30.04mgZn/g casein. Copyright © 2014 Elsevier B.V. All rights reserved.

Establishment of an inflamed animal model of diabetic nephropathy.

PubMed

Ma, Kun Ling; Zhang, Yang; Liu, Jing; Wu, Yu; Hu, Ze Bo; Ruan, Xiong Zhong; Liu, Bi Cheng

2014-01-01

Inflammatory stress plays a crucial role in the progression of diabetic nephropathy (DN). This study aimed to establish a novel inflamed animal model of DN and to evaluate its significance in DN. Nondiabetic db/m mice and diabetic db/db mice were randomly divided into four groups: db/m, db/m+casein, db/db, and db/db+casein for eight weeks. Casein was subcutaneously injected to induce chronic inflammation. Body weight and albumin to creatinine ratio (ACR) in the urine were measured every week. The plasma levels of serum amyloid protein A (SAA) and tumour necrotic factor-α (TNF-α) were determined with the enzyme-linked immunosorbent assay. The morphological changes to the renal pathology and ultra-microstructures were checked by pathological staining and electron microscopy. Immunofluorescent staining and Western blotting were used to determine the protein expression of podocyte-specific molecules and inflammatory cytokines in kidneys. ACR, plasma levels of SAA and TNF-α, protein expression of inflammatory cytokines, mesangial expansion, collagen accumulation, and foot process effacement in kidneys of casein-injected db/db mice were significantly increased compared with the db/db mice. Casein injection markedly decreased the protein expression of Wilms' tumor-1 and nephrin in kidneys of db/db mice, which are specific podocyte biomarkers, suggesting that chronic inflammation accelerates podocyte injuries in db/db mice. Interestingly, no obvious urinary protein, inflammatory cytokine expression, or histological changes in the kidneys of casein-injected db/m mice were found compared with the db/m mice. An inflamed animal model of DN was successfully established and may provide a useful tool for investigating the pathogenesis of DN under inflammatory stress.

Yeast casein kinase 2 governs morphology, biofilm formation, cell wall integrity, and host cell damage of Candida albicans

PubMed Central

Irrizary, Jihyun; Liboro, Karl; Bogarin, Thania; Macias, Marlene; Eivers, Edward; Porter, Edith; Filler, Scott G.

2017-01-01

The regulatory networks governing morphogenesis of a pleomorphic fungus, Candida albicans are extremely complex and remain to be completely elucidated. This study investigated the function of C. albicans yeast casein kinase 2 (CaYck2p). The yck2Δ/yck2Δ strain displayed constitutive pseudohyphae in both yeast and hyphal growth conditions, and formed enhanced biofilm under non-biofilm inducing condition. This finding was further supported by gene expression analysis of the yck2Δ/yck2Δ strain which showed significant upregulation of UME6, a key transcriptional regulator of hyphal transition and biofilm formation, and cell wall protein genes ALS3, HWP1, and SUN41, all of which are associated with morphogenesis and biofilm architecture. The yck2Δ/yck2Δ strain was hypersensitive to cell wall damaging agents and had increased compensatory chitin deposition in the cell wall accompanied by an upregulation of the expression of the chitin synthase genes, CHS2, CHS3, and CHS8. Absence of CaYck2p also affected fungal-host interaction; the yck2Δ/yck2Δ strain had significantly reduced ability to damage host cells. However, the yck2Δ/yck2Δ strain had wild-type susceptibility to cyclosporine and FK506, suggesting that CaYck2p functions independently from the Ca+/calcineurin pathway. Thus, in C. albicans, Yck2p is a multifunctional kinase that governs morphogenesis, biofilm formation, cell wall integrity, and host cell interactions. PMID:29107946

Disorder in milk proteins: caseins, intrinsically disordered colloids.

PubMed

Redwan, Elrashdy M; Xue, Bin; Almehdar, Hussein A; Uversky, Vladimir N

2015-01-01

This article opens a series of reviews on the abundance and roles of intrinsic disorder in milk proteins. The focus of this introductory article on caseins is symbolic, since caseins were among the first recognized functional unfolded proteins and since they are definitely the most disordered, the most abundant, and the most studied of all milk proteins. In eutherian milks, the casein family includes at least three and usually four major members (αs1-, αs2-, β-, and κ-caseins) that are unrelated in sequence. However, in some species, two different αS2-casein genes are active, and therefore the total number of caseins can be as high as five. These proteins have found a number of uses in food industry. The functional repertoire of caseins ranges from nutritional function to involvement in the improving and/or maintaining cardiovascular health, to crucial contribution to the milk capacity to transport calcium phosphate, to serve as molecular chaperones, and to protect the mother's mammary gland against amyloidoses and ectopic calcification. An intricate feature of caseins is their ability to assemble to colloidal protein particles, casein micelles, serving to sequester and transport amorphous calcium phosphate. These and many other functions of caseins are obviously dependent on their intrinsically disordered nature and are controlled by various posttranslational modifications. Since various aspects of casein structure and function are rather well studied and since several recent reviews emphasized the functional roles of caseins' intrinsic disorder, the major goal of this article is to show how intrinsic disorder is encoded in the amino acid sequences of these proteins.

Polymorphism in ovine ANXA9 gene and physic-chemical properties and the fraction of protein in milk.

PubMed

Pecka-Kiełb, Ewa; Czerniawska-Piątkowska, Ewa; Kowalewska-Łuczak, Inga; Vasil, Milan

2018-04-16

Annexin A9 (ANXA9) is a specific fatty acid transport protein. ANXA9 gene is expressed in various tissues, including secretory tissue and mammary glands. The association between three SNPs of the ANXA9 gene and sheep's milk compositions was assessed. Genotype analysis was performed with the use of PCR-RFLP method. The studied ANXA9 polymorphisms had the following MAF (Major Allele Frequency): SNP1: allele G 0,66; SNP2: allele G 0,54; SNP3: allele C 0,57. The study found the most desired profile of protein fractions, namely an increased kappa-casein fractions and a decreased level of whey protein in sheep's milk for SNP1 and SNP3 polymorphisms. Sheep with the SNP1 GA genotype had the highest (P <0.05) content of fat and dry matter in milk. AXNA9 gene polymorphism did not influence the levels of protein, lactose or urea in sheep's milk. The information contained in this study may be useful for determining the impact of the ANXA9 gene on sheep's milk. The ANXA9 SNP1 and SNP3 polymorphisms results could be included in the breeding programs to select the sheep with the genotypes ensuring the highest kappa-casein levels in milk. However, it is worth conducting further research on ANXA9 and milk composition in larger herds of animals and various breeds of sheep. This article is protected by copyright. All rights reserved.

Co-autodisplay of Z-domains and bovine caseins on the outer membrane of E. coli.

PubMed

Yoo, Gu; Saenger, Thorsten; Bong, Ji-Hong; Jose, Joachim; Kang, Min-Jung; Pyun, Jae-Chul

2015-12-01

In this work, two proteins, Z-domains and bovine casein, were auto-displayed on the outer membrane of the same Escherichia coli cells by co-transformation of two different auto-display vectors. On the basis of SDS-PAGE densitometry, Z-domains and bovine casein were expressed at 3.12 × 10⁵ and 1.55 × 10⁵ proteins/E. coli cell, respectively. The co-auto-displayed Z-domains had antibody-binding activity and the bovine casein had adhesive properties. E. coli with co-auto-displayed proteins were analyzed by fluorescence assisted cell sorting (FACS). E. coli with co-auto-displayed Z-domains and bovine casein aggregated due to hydrophobic interaction. For application to immunoassays, the Z-domain activity was estimated after (1) immobilizing the E. coli and (2) forming an OM layer. E. coli with co-auto-displayed two proteins that were immobilized on a polystyrene microplate had the same antibody-binding activity as did E. coli with auto-displayed Z-domains only. The OM layer from the co-transformed E. coli had Z-domains and bovine casein expressed at a 1:2 ratio from antibody-binding activity measurements. Copyright © 2015 Elsevier B.V. All rights reserved.

Stability of casein micelles in milk

NASA Astrophysics Data System (ADS)

Tuinier, R.; de Kruif, C. G.

2002-07-01

Casein micelles in milk are proteinaceous colloidal particles and are essential for the production of flocculated and gelled products such as yogurt, cheese, and ice-cream. The colloidal stability of casein micelles is described here by a calculation of the pair potential, containing the essential contributions of brush repulsion, electrostatic repulsion, and van der Waals attraction. The parameters required are taken from the literature. The results are expressed by the second osmotic virial coefficient and are quite consistent with experimental findings. It appears that the stability is mainly attributable to a steric layer of κ-casein, which can be described as a salted polyelectrolyte brush.

Genome wide gene expression analysis of the posterior capsule in patients with osteoarthritis and knee flexion contracture.

PubMed

Campbell, Thomas Mark; Trudel, Guy; Wong, Kayleigh Kristin; Laneuville, Odette

2014-11-01

Knee flexion contractures (KFC) are limitations in the ability to fully extend the knee joint. In people with knee osteoarthritis (OA), KFC are common, impair function, and worsen outcomes after arthroplasty. In KFC, the posterior knee capsule is believed to play a key role, but the pathophysiology remains poorly understood. We sought to identify gene expression differences in the posterior knee capsule of patients with OA with and without KFC. Capsule tissue was obtained from the knees of 12 subjects diagnosed with advanced-stage OA at the time of knee arthroplasty surgery. The presence or absence of KFC allocated patients into 2 groups using a case-control design. Genomewide capsular gene expression was compared between the 2 patient groups. Confirmation of differential expression of the corresponding proteins was performed by immunohistochemistry on tissue sections. There were no significant demographic differences between the patients with OA with KFC and without KFC save for reduced extension in their surgical knee (p<0.01). KFC patients showed a 6.4-fold decrease in CSN1S1 (p=0.017) gene expression and a 3.7-, 2.0-, and 2.6-fold increase in CHAD, Sox9, and Cyr61 gene expression, respectively (p=0.001, 0.004, 0.001, respectively). There were corresponding increases in protein levels for chondroadherin, sex determining region Y-box 9, and casein alphaS1 (all p<0.05). Functional analysis of the differentially expressed genes indicated a strong association with pathways related to the extracellular matrix and to tissue fibrosis. Posterior capsules in endstage OA knees with KFC exhibited differential expression of 4 genes all previously documented to be associated with tissue fibrosis.

Short communication: Tryptic β-casein hydrolysate modulates enteric nervous system development in primary culture.

PubMed

Cossais, F; Clawin-Rädecker, I; Lorenzen, P C; Klempt, M

2017-05-01

The intestinal tract of the newborn is particularly sensitive to gastrointestinal disorders, such as infantile diarrhea or necrotizing colitis. Perinatal development of the gut also encompasses the maturation of the enteric nervous system (ENS), a main regulator of intestinal motility and barrier functions. It was recently shown that ENS maturation can be enhanced by nutritional factors to improve intestinal maturation. Bioactivity of milk proteins is often latent, requiring the release of bioactive peptides from inactive native proteins. Several casein-derived hydrolysates presenting immunomodulatory properties have been described recently. Furthermore, accumulating data indicate that milk-derived hydrolysate can enhance gut maturation and enrichment of milk formula with such hydrolysates has recently been proposed. However, the capability of milk-derived bioactive hydrolysate to target ENS maturation has not been analyzed so far. We, therefore, investigated the potential of a recently described tryptic β-casein hydrolysate to modulate ENS growth parameters in an in vitro model of rat primary culture of ENS. Rat primary cultures of ENS were incubated with a bioactive tryptic β-casein hydrolysate and compared with untreated controls or to cultures treated with native β-casein or a Prolyve β-casein hydrolysate (Lyven, Colombelles, France). Differentiation of enteric neurons and enteric glial cells, and establishment of enteric neural network were analyzed using immunohistochemistry and quantitative PCR. Effect of tryptic β-casein hydrolysate on bone morphogenetic proteins (BMP)/Smad pathway, an essential regulator of ENS development, was further assessed using quantitative PCR and immunochemistry. Tryptic β-casein hydrolysate stimulated neurite outgrowth and simultaneously modulated the formation of enteric ganglia-like structures, whereas native β-casein or Prolyve β-casein hydrolysate did not. Additionally, treatment with tryptic bioactive β-casein hydrolysate increased the expression of the glial marker glial fibrillary acidic protein and induced profound modifications of enteric glial cells morphology. Finally, expression of BMP2 and BMP4 and activation of Smad1/5 was altered after treatment with tryptic bioactive β-casein hydrolysate. Our data suggests that this milk-derived bioactive hydrolysate modulates ENS maturation through the regulation of BMP/Smad-signaling pathway. This study supports the need for further investigation on the influence of milk-derived bioactive peptides on ENS and intestinal maturation in vivo. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

CK2(beta)tes gene encodes a testis-specific isoform of the regulatory subunit of casein kinase 2 in Drosophila melanogaster.

PubMed

Kalmykova, Alla I; Shevelyov, Yuri Y; Polesskaya, Oksana O; Dobritsa, Anna A; Evstafieva, Alexandra G; Boldyreff, Brigitte; Issinger, Olaf-Georg; Gvozdev, Vladimir A

2002-03-01

An earlier described CK2(beta)tes gene of Drosophila melanogaster is shown to encode a male germline specific isoform of regulatory beta subunit of casein kinase 2. Western-analysis using anti-CK2(beta)tes Ig revealed CK2(beta)tes protein in Drosophila testes extract. Expression of a CK2(beta)tes-beta-galactosidase fusion protein driven by the CK2(beta)tes promoter was found in transgenic flies at postmitotic stages of spermatogenesis. Examination of biochemical characteristics of a recombinant CK2(beta)tes protein expressed in Escherichia coli revealed properties similar to those of CK2beta: (a) CK2(beta)tes protein stimulates CK2alpha catalytic activity toward synthetic peptide; (b) it inhibits phosphorylation of calmodulin and mediates stimulation of CK2alpha by polylysine; (c) it is able to form (CK2(beta)tes)2 dimers, as well as (CK2alpha)2(CK2(beta)tes)2 tetramers. Using the yeast two-hybrid system and coimmunoprecipitation analysis of protein extract from Drosophila testes, we demonstrated an association between CK2(beta)tes and CK2alpha. Northern-analysis has shown that another regulatory (beta') subunit found recently in D. melanogaster genome is also testis-specific. Thus, we describe the first example of two tissue-specific regulatory subunits of CK2 which might serve to provide CK2 substrate recognition during spermatogenesis.

Characterization of casein and alpha lactalbumin of African elephant (Loxodonta africana) milk.

PubMed

Madende, M; Osthoff, G; Patterton, H-G; Patterton, H E; Martin, P; Opperman, D J

2015-12-01

The current research reports partial characterization of the caseins and α-lactalbumin (α-LA) of the African elephant with proposed unique structure-function properties. Extensive research has been carried out to understand the structure of the casein micelles. Crystallographic structure elucidation of caseins and casein micelles is not possible. Consequently, several models have been developed in an effort to describe the casein micelle, specifically of cow milk. Here we report the characterization of African elephant milk caseins. The κ-caseins and β-caseins were investigated, and their relative ratio was found to be approximately 1:8.5, whereas α-caseins were not detected. The gene sequence of β-casein in the NCBI database was revisited, and a different sequence in the N-terminal region is proposed. Amino acid sequence alignment and hydropathy plots showed that the κ-casein of African elephant milk is similar to that of other mammals, whereas the β-casein is similar to the human protein, and displayed a section of unique AA composition and additional hydrophilic regions compared with bovine caseins. Elephant milk is destabilized by 62% alcohol, and it is speculated that the β-casein characteristics may allow maintenance of the colloidal nature of the casein micelle, a role that was previously only associated with κ-casein. The oligosaccharide content of milk was reported to be low in dairy animals but high in some other species such as humans and elephants. In the milk of the African elephant, lactose and oligosaccharides both occur at high levels. These levels are typically related to the content of α-LA in the mammary gland and thus point to a specialized carbohydrate synthesis, where the whey protein α-LA plays a role. We report the characterization of African elephant α-LA. Homology modeling of the α-LA showed that it is structurally similar to crystal structures of other mammalian species, which in turn may be an indication that its functional properties, such as lactose synthesis, should not be impaired. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Quantity and source of dietary protein influence metabolite production by gut microbiota and rectal mucosa gene expression: a randomized, parallel, double-blind trial in overweight humans.

PubMed

Beaumont, Martin; Portune, Kevin Joseph; Steuer, Nils; Lan, Annaïg; Cerrudo, Victor; Audebert, Marc; Dumont, Florent; Mancano, Giulia; Khodorova, Nadezda; Andriamihaja, Mireille; Airinei, Gheorghe; Tomé, Daniel; Benamouzig, Robert; Davila, Anne-Marie; Claus, Sandrine Paule; Sanz, Yolanda; Blachier, François

2017-10-01

Background: Although high-protein diets (HPDs) are frequently consumed for body-weight control, little is known about the consequences for gut microbiota composition and metabolic activity and for large intestine mucosal homeostasis. Moreover, the effects of HPDs according to the source of protein need to be considered in this context. Objective: The objective of this study was to evaluate the effects of the quantity and source of dietary protein on microbiota composition, bacterial metabolite production, and consequences for the large intestinal mucosa in humans. Design: A randomized, double-blind, parallel-design trial was conducted in 38 overweight individuals who received a 3-wk isocaloric supplementation with casein, soy protein, or maltodextrin as a control. Fecal and rectal biopsy-associated microbiota composition was analyzed by 16S ribosomal DNA sequencing. Fecal, urinary, and plasma metabolomes were assessed by 1 H-nuclear magnetic resonance. Mucosal transcriptome in rectal biopsies was determined with the use of microarrays. Results: HPDs did not alter the microbiota composition, but induced a shift in bacterial metabolism toward amino acid degradation with different metabolite profiles according to the protein source. Correlation analysis identified new potential bacterial taxa involved in amino acid degradation. Fecal water cytotoxicity was not modified by HPDs, but was associated with a specific microbiota and bacterial metabolite profile. Casein and soy protein HPDs did not induce inflammation, but differentially modified the expression of genes playing key roles in homeostatic processes in rectal mucosa, such as cell cycle or cell death. Conclusions: This human intervention study shows that the quantity and source of dietary proteins act as regulators of gut microbiota metabolite production and host gene expression in the rectal mucosa, raising new questions on the impact of HPDs on the large intestine mucosa homeostasis. This trial was registered at clinicaltrials.gov as NCT02351297. © 2017 American Society for Nutrition.

Short communication: Genetic parameters for milk protein composition predicted using mid-infrared spectroscopy in the French Montbéliarde, Normande, and Holstein dairy cattle breeds.

PubMed

Sanchez, M P; Ferrand, M; Gelé, M; Pourchet, D; Miranda, G; Martin, P; Brochard, M; Boichard, D

2017-08-01

Genetic parameters for the major milk proteins were estimated in the 3 main French dairy cattle breeds (i.e. Montbéliarde, Normande, and Holstein) as part of the PhénoFinlait program. The 6 major milk protein contents as well as the total protein content (PC) were estimated from mid-infrared spectrometry on 133,592 test-day milk samples from 20,434 cows in first lactation. Lactation means, expressed as a percentage of milk (protein contents) or of protein (protein fractions), were analyzed with an animal mixed model including fixed environmental effects (herd, year × month of calving, and spectrometer) and a random genetic effect. Genetic parameter estimates were very consistent across breeds. Heritability estimates (h 2 ) were generally higher for protein fractions than for protein contents. They were moderate to high for α S1 -casein, α S2 -casein, β-casein, κ-casein, and α-lactalbumin (0.25 < h 2 < 0.72). In each breed, β-lactoglobulin was the most heritable trait (0.61 < h 2 < 0.86). Genetic correlations (r g ) varied depending on how the percentage was expressed. The PC was strongly positively correlated with protein contents but almost genetically independent from protein fractions. Protein fractions were generally in opposition, except between κ-casein and α-lactalbumin (0.39 < r g < 0.46) and κ-casein and α S2 -casein (0.36 < r g < 0.49). Between protein contents, r g estimates were positive, with highest values found between caseins (0.83 < r g < 0.98). In the 3 breeds, β-lactoglobulin was negatively correlated with caseins (-0.75 < r g < -0.08), in particular with κ-casein (-0.75 < r g < -0.55). These results, obtained from a large panel of cows of the 3 main French dairy cattle breeds, show that routinely collected mid-infrared spectra could be used to modify milk protein composition by selection. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Selection for genetic markers in beef cattle reveals complex associations of thyroglobulin and casein1-S1 with carcass and meat traits

USDA-ARS?s Scientific Manuscript database

Genetic markers in casein (CSN1S1) and thyroglobulin (TG) genes have previously been associated with fat distribution in cattle. Determining the nature of these genetic associations (additive, recessive, or dominant) has been difficult because both markers have small minor allele frequencies in mos...

Effect of the combinations between pea proteins and soluble fibres on cholesterolaemia and cholesterol metabolism in rats.

PubMed

Parolini, Cinzia; Manzini, Stefano; Busnelli, Marco; Rigamonti, Elena; Marchesi, Marta; Diani, Erika; Sirtori, Cesare R; Chiesa, Giulia

2013-10-01

Many functional foods and dietary supplements have been reported to be beneficial for the management of dyslipidaemia, one of the major risk factors for CVD. Soluble fibres and legume proteins are known to be a safe and practical approach for cholesterol reduction. The present study aimed at investigating the hypocholesterolaemic effect of the combinations of these bioactive vegetable ingredients and their possible effects on the expression of genes regulating cholesterol homeostasis. A total of six groups of twelve rats each were fed, for 28 d, Nath's hypercholesterolaemic diets, differing in protein and fibre sources, being, respectively, casein and cellulose (control), pea proteins and cellulose (pea), casein and oat fibres (oat), casein and apple pectin (pectin), pea proteins and oat fibres (pea+oat) and pea proteins and apple pectin (pea+pectin). Administration of each vegetable-containing diet was associated with lower total cholesterol concentrations compared with the control. The combinations (pea+oat and pea+pectin) were more efficacious than fibres alone in modulating cholesterolaemia ( - 53 and - 54%, respectively, at 28 d; P< 0·005). In rats fed the diets containing oat fibres or apple pectin, alone or in combination with pea proteins, a lower hepatic cholesterol content (P< 0·005) and higher hepatic mRNA concentrations of CYP7A1 and NTCP were found when compared with the control rats (P< 0·05). In summary, the dietary combinations of pea proteins and oat fibres or apple pectin are extremely effective in lowering plasma cholesterol concentrations in rats and affect cellular cholesterol homeostasis by up-regulating genes involved in hepatic cholesterol turnover.

Milk Intolerance, Beta-Casein and Lactose.

PubMed

Pal, Sebely; Woodford, Keith; Kukuljan, Sonja; Ho, Suleen

2015-08-31

True lactose intolerance (symptoms stemming from lactose malabsorption) is less common than is widely perceived, and should be viewed as just one potential cause of cows' milk intolerance. There is increasing evidence that A1 beta-casein, a protein produced by a major proportion of European-origin cattle but not purebred Asian or African cattle, is also associated with cows' milk intolerance. In humans, digestion of bovine A1 beta-casein, but not the alternative A2 beta-casein, releases beta-casomorphin-7, which activates μ-opioid receptors expressed throughout the gastrointestinal tract and body. Studies in rodents show that milk containing A1 beta-casein significantly increases gastrointestinal transit time, production of dipeptidyl peptidase-4 and the inflammatory marker myeloperoxidase compared with milk containing A2 beta-casein. Co-administration of the opioid receptor antagonist naloxone blocks the myeloperoxidase and gastrointestinal motility effects, indicating opioid signaling pathway involvement. In humans, a double-blind, randomized cross-over study showed that participants consuming A1 beta-casein type cows' milk experienced statistically significantly higher Bristol stool values compared with those receiving A2 beta-casein milk. Additionally, a statistically significant positive association between abdominal pain and stool consistency was observed when participants consumed the A1 but not the A2 diet. Further studies of the role of A1 beta-casein in milk intolerance are needed.

Milk Intolerance, Beta-Casein and Lactose

PubMed Central

Pal, Sebely; Woodford, Keith; Kukuljan, Sonja; Ho, Suleen

2015-01-01

True lactose intolerance (symptoms stemming from lactose malabsorption) is less common than is widely perceived, and should be viewed as just one potential cause of cows’ milk intolerance. There is increasing evidence that A1 beta-casein, a protein produced by a major proportion of European-origin cattle but not purebred Asian or African cattle, is also associated with cows’ milk intolerance. In humans, digestion of bovine A1 beta-casein, but not the alternative A2 beta-casein, releases beta-casomorphin-7, which activates μ-opioid receptors expressed throughout the gastrointestinal tract and body. Studies in rodents show that milk containing A1 beta-casein significantly increases gastrointestinal transit time, production of dipeptidyl peptidase-4 and the inflammatory marker myeloperoxidase compared with milk containing A2 beta-casein. Co-administration of the opioid receptor antagonist naloxone blocks the myeloperoxidase and gastrointestinal motility effects, indicating opioid signaling pathway involvement. In humans, a double-blind, randomized cross-over study showed that participants consuming A1 beta-casein type cows’ milk experienced statistically significantly higher Bristol stool values compared with those receiving A2 beta-casein milk. Additionally, a statistically significant positive association between abdominal pain and stool consistency was observed when participants consumed the A1 but not the A2 diet. Further studies of the role of A1 beta-casein in milk intolerance are needed. PMID:26404362

Cloning, characterization, expression and antifungal activity of an alkaline serine protease of Aureobasidium pullulans PL5 involved in the biological control of postharvest pathogens.

PubMed

Zhang, Dianpeng; Spadaro, Davide; Valente, Silvia; Garibaldi, Angelo; Gullino, Maria Lodovica

2012-02-15

An alkaline protease gene was amplified from genomic DNA and cDNA of the antagonistic yeast-like fungus Aureobasidium pullulans PL5, a biocontrol agent effective against Monilinia laxa on stone fruit and Botrytis cinerea and Penicillium expansum on pome fruits. An open reading frame of 1248 bp encoding a 415-amino acid (aa) protein with a calculated molecular weight (M(r)) of 42.9 kDa and an isoelectric point (pI) of 4.5 was characterized. The cDNAALP5 gene had an 18-amino acid signal peptide, one N-gylcosylation, one histidine active site, and one serine active site. The ALP5 gene with a M(r) of 1351 bp contained two introns. One intron was of 54 bp, while the other was of 50 bp. Protein BLAST and phylogenetic tree analysis of the deduced amino sequences from the cDNAALP5 gene showed that the encoded protein had 100% homology to a protease enzyme (ALP2) of a sea strain of A. pullulans, suggesting that the protein ALP5 was an alkaline serine protease. Expression of ALP5 in Escherichia coli BL21 (DE3), followed by identification with Western-blotting, purification with Ni-NTA and analysis of enzymatic activity, yielded an homogeneous recombinant ALP5 which hydrolysed the substrate casein and inhibited the mycelial growth of the pathogens. At its optimal pH of 10.0 and reaction temperature of 50°C, the recombinant protease exhibited the highest activity towards the substrate casein, though the highest stability was at lower temperatures and pH between 7.0 and 9.0. This study provided the direct evidence that extracellular proteases secreted by the antagonist A. pullulans PL5 played a role in the biocontrol activities against some postharvest pathogens of apple and peach. Copyright © 2011 Elsevier B.V. All rights reserved.

Hormonal regulation of platypus Beta-lactoglobulin and monotreme lactation protein genes.

PubMed

Enjapoori, Ashwantha Kumar; Lefèvre, Christophe M; Nicholas, Kevin R; Sharp, Julie A

2017-02-01

Endocrine regulation of milk protein gene expression in marsupials and eutherians is well studied. However, the evolution of this complex regulation that began with monotremes is unknown. Monotremes represent the oldest lineage of extant mammals and the endocrine regulation of lactation in these mammals has not been investigated. Here we characterised the proximal promoter and hormonal regulation of two platypus milk protein genes, Beta-lactoglobulin (BLG), a whey protein and monotreme lactation protein (MLP), a monotreme specific milk protein, using in vitro reporter assays and a bovine mammary epithelial cell line (BME-UV1). Insulin and dexamethasone alone provided partial induction of MLP, while the combination of insulin, dexamethasone and prolactin was required for maximal induction. Partial induction of BLG was achieved by insulin, dexamethasone and prolactin alone, with maximal induction using all three hormones. Platypus MLP and BLG core promoter regions comprised transcription factor binding sites (e.g. STAT5, NF-1 and C/EBPα) that were conserved in marsupial and eutherian lineages that regulate caseins and whey protein gene expression. Our analysis suggests that insulin, dexamethasone and/or prolactin alone can regulate the platypus MLP and BLG gene expression, unlike those of therian lineage. The induction of platypus milk protein genes by lactogenic hormones suggests they originated before the divergence of marsupial and eutherians. Copyright © 2015 Elsevier Inc. All rights reserved.

Identification of alleles and genotypes of beta-casein with DNA sequencing analysis in Chinese Holstein cow.

PubMed

Dai, Ronghua; Fang, Yu; Zhao, Wenjing; Liu, Shuyun; Ding, Jinmei; Xu, Ke; Yang, Lingyu; He, Chuan; Ding, Fangmei; Meng, He

2016-08-01

The study reported in this Regional Research Communication aimed to analyse the genetic polymorphisms of β-casein in Chinese Holstein cows. β-casein has received considerable research interest in the dairy industry and animal breeding in recent years as a source not only of high quality protein, but also of bioactive peptides that may be linked to health effects. Morever, the polymorphic nature of β-casein and its association with milk production traits, composition, and quality also attracted several efforts in evaluating the allelic distribution of β-casein locus as a potential dairy trait marker. However, few data on beta-casein variants are available for the Chinese Holstein cow. In the present paper, one hundred and thirty three Holstein cows were included in the analysis. Results revealed the presence of 5 variants (A1, A2, A3, B and I), preponderance of the genotype A1A2 (0·353) and superiorities of A1/A2 alleles (0·432 and 0·459, respectively) in the population. Sequence analysis of β-casein gene in the cows showed four nucleotide changes in exon 7. Our study can provide reference and guidance for selection for superior milk for industrial applications and crossbreeding and genetic improvement programmes.

Geographic distribution of haplotype diversity at the bovine casein locus

PubMed Central

Jann, Oliver C; Ibeagha-Awemu, Eveline M; Özbeyaz, Ceyhan; Zaragoza, Pilar; Williams, John L; Ajmone-Marsan, Paolo; Lenstra, Johannes A; Moazami-Goudarzi, Katy; Erhardt, Georg

2004-01-01

The genetic diversity of the casein locus in cattle was studied on the basis of haplotype analysis. Consideration of recently described genetic variants of the casein genes which to date have not been the subject of diversity studies, allowed the identification of new haplotypes. Genotyping of 30 cattle breeds from four continents revealed a geographically associated distribution of haplotypes, mainly defined by frequencies of alleles at CSN1S1 and CSN3. The genetic diversity within taurine breeds in Europe was found to decrease significantly from the south to the north and from the east to the west. Such geographic patterns of cattle genetic variation at the casein locus may be a result of the domestication process of modern cattle as well as geographically differentiated natural or artificial selection. The comparison of African Bos taurus and Bos indicus breeds allowed the identification of several Bos indicus specific haplotypes (CSN1S1*C-CSN2*A2-CSN3*AI/CSN3*H) that are not found in pure taurine breeds. The occurrence of such haplotypes in southern European breeds also suggests that an introgression of indicine genes into taurine breeds could have contributed to the distribution of the genetic variation observed. PMID:15040901

Identification and temporal expression of putative circadian clock transcripts in the amphipod crustacean Talitrus saltator

PubMed Central

O’Grady, Joseph F.; Hoelters, Laura S.; Swain, Martin T.

2016-01-01

Background Talitrus saltator is an amphipod crustacean that inhabits the supralittoral zone on sandy beaches in the Northeast Atlantic and Mediterranean. T. saltator exhibits endogenous locomotor activity rhythms and time-compensated sun and moon orientation, both of which necessitate at least one chronometric mechanism. Whilst their behaviour is well studied, currently there are no descriptions of the underlying molecular components of a biological clock in this animal, and very few in other crustacean species. Methods We harvested brain tissue from animals expressing robust circadian activity rhythms and used homology cloning and Illumina RNAseq approaches to sequence and identify the core circadian clock and clock-related genes in these samples. We assessed the temporal expression of these genes in time-course samples from rhythmic animals using RNAseq. Results We identified a comprehensive suite of circadian clock gene homologues in T. saltator including the ‘core’ clock genes period (Talper), cryptochrome 2 (Talcry2), timeless (Taltim), clock (Talclk), and bmal1 (Talbmal1). In addition we describe the sequence and putative structures of 23 clock-associated genes including two unusual, extended isoforms of pigment dispersing hormone (Talpdh). We examined time-course RNAseq expression data, derived from tissues harvested from behaviourally rhythmic animals, to reveal rhythmic expression of these genes with approximately circadian period in Talper and Talbmal1. Of the clock-related genes, casein kinase IIβ (TalckIIβ), ebony (Talebony), jetlag (Taljetlag), pigment dispensing hormone (Talpdh), protein phosphatase 1 (Talpp1), shaggy (Talshaggy), sirt1 (Talsirt1), sirt7 (Talsirt7) and supernumerary limbs (Talslimb) show temporal changes in expression. Discussion We report the sequences of principle genes that comprise the circadian clock of T. saltator and highlight the conserved structural and functional domains of their deduced cognate proteins. Our sequencing data contribute to the growing inventory of described comparative clocks. Expression profiling of the identified clock genes illuminates tantalising targets for experimental manipulation to elucidate the molecular and cellular control of clock-driven phenotypes in this crustacean. PMID:27761341

A Metabolic Trade-Off Modulates Policing of Social Cheaters in Populations of Pseudomonas aeruginosa

PubMed Central

Yan, Huicong; Wang, Meizhen; Sun, Feng; Dandekar, Ajai A.; Shen, Dongsheng; Li, Na

2018-01-01

Pseudomonas aeruginosa uses quorum sensing (QS) to regulate the production of public goods such as the secreted protease elastase. P. aeruginosa requires the LasI–LasR QS circuit to induce elastase and enable growth on casein as the sole carbon and energy source. The LasI–LasR system also induces a second QS circuit, the RhlI–RhlR system. During growth on casein, LasR-mutant social cheaters emerge, and this can lead to a population collapse. In a minimal medium containing ammonium sulfate as a nitrogen source, populations do not collapse, and cheaters and cooperators reach a stable equilibrium; however, without ammonium sulfate, cheaters overtake the cooperators and populations collapse. We show that ammonium sulfate enhances the activity of the RhlI–RhlR system in casein medium and this leads to increased production of cyanide, which serves to control levels of cheaters. This enhancement of cyanide production occurs because of a trade-off in the metabolism of glycine: exogenous ammonium ion inhibits the transformation of glycine to 5,10-methylenetetrahydrofolate through a reduction in the expression of the glycine cleavage genes gcvP1 and gcvP2, thereby increasing the availability of glycine as a substrate for RhlR-regulated hydrogen cyanide synthesis. Thus, environmental ammonia enhances cyanide production and stabilizes QS in populations of P. aeruginosa. PMID:29535700

Release of β-casomorphins 5 and 7 during simulated gastro-intestinal digestion of bovine β-casein variants and milk-based infant formulas.

PubMed

Noni, Ivano De

2008-10-15

The release of β-casomorphin-5 (BCM5) and β-casomorphin-7 (BCM7) was investigated during simulated gastro-intestinal digestion (SGID) of bovine β-casein variants (n=3), commercial milk-based infant formulas (n=6) and experimental infant formulas (n=3). SGID included pepsin digestion at pH 2.0, 3.0 and 4.0 and further hydrolysis with Corolase PP™. β-Casein (β-CN) variants were extracted from raw milks coming from cows of Holstein-Friesian and Jersey breeds. Genomic DNA was isolated from milk and the β-CN genotype was determined by a PCR-based method. Phenotype at protein level was determined by capillary zone electrophoresis in order to ascertain the level of gene expression. Recognition and quantification of BCMs involved HPLC coupled to tandem MS. Regardless of the pH, BCM7 generated from variants A1 and B of β-CN (5-176mmol/mol casein) the highest amount being released during SGID of form B. As expected, the peptide was not released from variant A2 at any steps of SGID. BCM5 was not formed in hydrolysates irrespective of either the genetic variant or the pH value during SGID. Variants A1, A2 and B of β-CN were present in all the commercial infant formulae (IFs) submitted to SGID. Accordingly, 16-297nmol BCM7 were released from 800ml IF, i.e. the daily recommended intake for infant. Industrial indirect-UHT treatments (156°C×6-9s) did not modify release of BCM7 and, during SGID, comparable peptide amounts formed in raw formulation and final heat-treated IFs. Copyright © 2008 Elsevier Ltd. All rights reserved.

Characterization of casein gene complex and genetic diversity analysis in Indian goats.

PubMed

Rout, P K; Kumar, A; Mandal, A; Laloe, D; Singh, S K; Roy, R

2010-04-01

Milk protein polymorphism plays an important role in genetic diversity analysis, phylogenetic studies, establishing geographical diversity, conservation decision, and improving breeding goals. Milk protein polymorphism in Indian goat breeds has not been well studied; therefore, an investigation was carried out to analyze the genetic structure of the casein gene and milk protein diversity at six milk protein loci in nine Indian goat breeds/genetic groups from varied agro-climatic zones. Milk protein genotyping was carried out in 1098 individual milk samples by SDS-PAGE at alphaS1-CN (CSN1S1), beta-CN (CSN2), alphaS2-CN (CSN1S2), kappa-CN (CSN3), beta-LG, and alpha-LA loci. Indian goats exhibited alphaS1-casein A allele in higher frequency in the majority of breeds except Ganjam and local goats. The alphaS1-casein A allele frequencies varied from 0.45 to 0.77. A total of 16 casein haplotypes were observed in seven breeds and breed specific haplotypes were observed with respect to geographic region. The average number of alleles was lowest in Ganjam (1.66 +/- 0.81) and highest in Sirohi goats (2.50 +/- 1.05). Expected heterozygosity at six different loci demonstrated genetic diversity and breed fragmentation. Neighbor-Joining tree was built basing on Nei's distance. There was about 16.95% variability due to differences between breeds, indicating a strong subdivision. Principal component analysis was carried out to highlight the relationship among breeds. The variability among goat breeds was contributed by alphaS2-CN, beta-LG and alphaS1-CN. The Indian goats exhibited alphaS1-CN (CSN1S1) A allele in higher frequency in all the breeds indicating the higher casein yield in their milk.

Phosphorylation and nuclear localization of the varicella-zoster virus gene 63 protein.

PubMed Central

Stevenson, D; Xue, M; Hay, J; Ruyechan, W T

1996-01-01

The protein encoded by varicella-zoster virus open reading frame 63 and carboxy-terminal deletions of the same were expressed either as fusion proteins at the carboxy terminus of the maltose-binding protein in Escherichia coli or independently in transfected mammalian cells. The truncations contained amino acids 1 to 142 (63 delta N) or 1 to 210 (63 delta K) of the complete 278-amino-acid primary sequence. Recombinant casein kinase II phosphorylated the 63F and 63 delta KF fusion proteins in vitro but did not phosphorylate the 63 delta NF fusion protein, implying that phosphorylation occurred between amino acids 142 and 210. Immunoprecipitation of 35S- or 32P-labelled extracts of cells transfected with plasmids expressing 63, 63 delta N, or 63 delta K also indicated that in situ phosphorylation most likely occurred between amino acids 142 and 210. These combined results suggest that casein kinase II plays a significant role in the phosphorylation of the varicella-zoster virus 63 protein. Indirect immunofluorescence of transfected cells indicated nuclear localization of the 63 protein and cytoplasmic localization of 63 delta K and 63 delta N, implying a requirement for sequences between amino acids 210 and 278 for efficient nuclear localization. PMID:8523589

Regulation of glycolysis in Kluyveromyces lactis: role of KlGCR1 and KlGCR2 in glucose uptake and catabolism.

PubMed

Neil, H; Lemaire, M; Wésolowski-Louvel, M

2004-03-01

In Kluyveromyces lactis, the casein kinase I (Rag8p) regulates the transcription of glycolytic genes and the expression of the low-affinity glucose transporter gene RAG1. This control involves the transcription factor Sck1p, a homologue of Sgc1p of Saccharomyces cerevisiae. SGC1 is known to interact genetically with ScGCR1 and ScGCR2, which code for regulators of glycolytic gene expression. Therefore, we studied the role of KlGCR1 and KlGCR2 genes in K. lactis. The Klgcr1 null mutant could not grow on glucose when respiration was blocked by antimycin A (Rag(- )phenotype). In contrast, the Klgcr2 null mutant could grow under the same conditions, although at a reduced rate. In both mutants, the transcription of glycolytic genes was affected, while that of ribosomal protein genes was not modified. Furthermore, the transcription of the glucose permease genes was also found to be affected in the two mutants, although dissimilarly. While RAG1 transcription decreased at high glucose concentrations, the expression of the high-affinity glucose permease gene HGT1 was unexpectedly impaired under gluconeogenic conditions, in the absence of glucose. Gel mobility shift assays performed with purified maltose-binding protein-KlGcr1p showed that KlGcr1p could interact directly with the promoters of the glycolytic genes, but not with the promoters of the glucose permease genes. Thus, the control exerted by KlGcr1p and KlGcr2p upon glucose transporter genes is probably indirect.

Gene expression and adiposity are modified by soy protein in male Zucker diabetic fatty rats.

PubMed

Banz, William J; Davis, Jeremy; Peterson, Richard; Iqbal, Muhammad J

2004-12-01

It has earlier been demonstrated that soy protein diets ameliorate the diabetic phenotype in obese Zucker rats. In this study, we further investigated physiological changes related to adiposity in male Zucker diabetic fatty rats consuming soy-based diets and compared these diets with the insulin-sensitizing drug, rosiglitazone. Transcript abundance of known genes was assessed in the livers to identify potential molecular connections between soy diets and adiposity. Male Zucker diabetic fatty rats were assigned to casein (C) protein, low-isoflavone soy (LIS) protein, high-isoflavone soy (HIS) protein, or C + rosiglitazone (CR) diets. Compared with the C diet, the LIS diet decreased plasma lipids and increased body weight, but did not change liver weight or carcass adiposity. HIS decreased plasma lipids, liver weight, and body weight. CR decreased plasma lipids and increased carcass adiposity and body weight with no effect on liver weight. In LIS livers, 15 genes involved in signaling and lipid metabolism were up-regulated 2-fold or higher. In HIS livers, seven genes had a 2-fold or higher change in abundance. However, in CR livers, none of the genes was significantly changed compared with the C diet. There appears to be a distinct change in gene expression associated with soy diets as compared with C-based diets and rosiglitazone treatment.

Mycobacterium-Inducible Nramp in Striped Bass (Morone saxatilis)

USGS Publications Warehouse

Burge, E.J.; Gauthier, David T.; Ottinger, C.A.; Van Veld, P.A.

2004-01-01

In mammals, the natural resistance-associated macrophage protein 1 gene, Nramp1, plays a major role in resistance to mycobacterial infections. Chesapeake Bay striped bass (Morone saxatilis) is currently experiencing an epizootic of mycobacteriosis that threatens the health of this ecologically and economically important species. In the present study, we characterized an Nramp gene in this species and obtained evidence that there is induction following Mycobacterium exposure. The striped bass Nramp gene (MsNramp) and a 554-amino-acid sequence contain all the signal features of the Nramp family, including a topology of 12 transmembrane domains (TM), the transport protein-specific binding-protein-dependent transport system inner membrane component signature, three N-linked glycosylation sites between TM 7 and TM 8, sites of casein kinase and protein kinase C phosphorylation in the amino and carboxy termini, and a tyrosine kinase phosphorylation site between TM 6 and TM 7. Phylogenetic analysis most closely grouped MsNramp with other teleost Nramp genes and revealed high sequence similarity with mammalian Nramp2. MsNramp expression was present in all tissues assayed by reverse transcription-PCR. Within 1 day of injection of Mycobacterium marinum, MsNramp expression was highly induced (17-fold higher) in peritoneal exudate (PE) cells compared to the expression in controls. The levels of MsNramp were three- and sixfold higher on days 3 and 15, respectively. Injection of Mycobacterium shottsii resulted in two-, five-, and threefold increases in gene expression in PE cells over the time course. This report is the first report of induction of an Nramp gene by mycobacteria in a poikilothermic vertebrate.

Effects of dietary glutamine on inflammatory mediator gene expressions in rats with streptozotocin-induced diabetes.

PubMed

Tsai, Pei-Hsuan; Yeh, Chui-Li; Liu, Jun-Jen; Chiu, Wan-Chun; Yeh, Sung-Ling

2012-03-01

This study investigated the effects of glutamine (Gln) supplementation on gene expressions of inflammatory mediators and cytokines associated with T-helper cell type 17 (Th17) regulation in diabetic rats. There were one normal control group and two diabetic groups in this study. Rats in the normal control group were fed a regular chow diet. One diabetic group (DM) was fed a common semipurified diet, and the other diabetic group received a diet in which part of the casein was replaced by Gln (DM-Gln), which provided 25% of the total amino acid nitrogen for 8 wk. Diabetes was induced by an intraperitoneal injection of nicotinamide followed by streptozotocin. Rats with blood glucose levels exceeding 200 mg/dL were considered diabetic. Blood samples and blood mononuclear cells of the animals were collected at the end of the study for further analysis. Gene expressions of transforming growth factor-β1 and interleukin-17A did not differ in blood mononuclear cells among the three groups. Expressions of interleukin-6, interleukin-23, monocyte chemotactic protein-1, and the receptor of the advanced glycated endproducts gene were higher in blood mononuclear cells and the ratio of reduced to oxidized glutathione was lower in erythrocytes in the DM group than in the normal control group. Messenger RNA expressions of these genes were lower, whereas the ratio of reduced to oxidized glutathione was higher in the DM-Gln group than in the DM group. Supplemental dietary Gln increased the antioxidant potential and downregulated the expressions of inflammatory mediators. However, Th17 might not be an important involved pathway and the regulatory effect of Gln on Th17 immune response was not obvious in this animal model. Copyright © 2012 Elsevier Inc. All rights reserved.

Fish protein intake induces fast-muscle hypertrophy and reduces liver lipids and serum glucose levels in rats.

PubMed

Kawabata, Fuminori; Mizushige, Takafumi; Uozumi, Keisuke; Hayamizu, Kohsuke; Han, Li; Tsuji, Tomoko; Kishida, Taro

2015-01-01

In our previous study, fish protein was proven to reduce serum lipids and body fat accumulation by skeletal muscle hypertrophy and enhancing basal energy expenditure in rats. In the present study, we examined the precise effects of fish protein intake on different skeletal muscle fiber types and metabolic gene expression of the muscle. Fish protein increased fast-twitch muscle weight, reduced liver triglycerides and serum glucose levels, compared with the casein diet after 6 or 8 weeks of feeding. Furthermore, fish protein upregulated the gene expressions of a fast-twitch muscle-type marker and a glucose transporter in the muscle. These results suggest that fish protein induces fast-muscle hypertrophy, and the enhancement of basal energy expenditure by muscle hypertrophy and the increase in muscle glucose uptake reduced liver lipids and serum glucose levels. The present results also imply that fish protein intake causes a slow-to-fast shift in muscle fiber type.

Factors contributing to the resistivity of a higher casein diet against choline deficiency-induced hyperhomocysteinemia in rats.

PubMed

Liu, Yi-qun; Liu, Ying; Morita, Tatsuya; Mori, Makoto; Sugiyama, Kimio

2012-01-01

The mechanism by which feeding a higher casein diet results in resistance to choline deprivation-induced hyperhomocysteinemia was investigated in rats. Plasma homocysteine concentration was significantly lower in rats fed a 30% casein diet (30C) than in rats fed a 10% casein diet (10C). Choline deprivation did not enhance plasma homocysteine concentration in rats fed 30C, while it significantly enhanced plasma homocysteine concentration in rats fed 10C. The choline deprivation-induced enhancement of plasma homocysteine concentration in rats fed 10C was significantly suppressed by methionine supplementation in a dose-dependent manner in the range of 0.1 to 0.3%, but the suppressive effect of methionine became smaller with an increase in supplementation level in the range of 0.3 to 0.5%. At a 0.5% supplementation level, methionine did not exhibit any suppressive effect on choline deprivation-induced hyperhomocysteinemia. The higher plasma homocysteine concentration in rats fed choline-deprived 10C+0.5% methionine was significantly decreased by concurrent supplementation with 0.32% glycine+0.94% serine to the level of rats fed 10C. Raising dietary total amino acid level by adding 3.61% branched-chain amino acids (BCAA)+4.5% acidic amino acids (AAA) to choline-deprived 10C+0.5% methionine+0.32% glycine+0.94% serine resulted in a further decrease in plasma homocysteine concentration to a level lower than the level in rats fed 10C. Choline deprivation-induced increases in hepatic S-adenosylhomocysteine and homocysteine concentrations were significantly suppressed by supplementation with glycine+serine and further suppressed by BCAA+AAA. Hepatic cystathionine β-synthase activity and its gene expression were significantly increased by BCAA+AAA. Hepatic triglyceride concentration changed in a manner similar to that of plasma homocysteine concentration. The results indicate that there are at least three factors contributing to the resistivity of rats fed a higher casein diet (30C) to choline deprivation-induced hyperhomocysteinemia, i.e., higher intake of methionine, higher intake of glycine and serine, and higher intake of other amino acids such as BCAA and AAA.

A fish protein hydrolysate alters fatty acid composition in liver and adipose tissue and increases plasma carnitine levels in a mouse model of chronic inflammation.

PubMed

Bjørndal, Bodil; Berge, Christ; Ramsvik, Marie Sannes; Svardal, Asbjørn; Bohov, Pavol; Skorve, Jon; Berge, Rolf K

2013-10-07

There is growing evidence that fish protein hydrolysate (FPH) diets affect mitochondrial fatty acid metabolism in animals. The aim of the study was to determine if FPH could influence fatty acid metabolism and inflammation in transgene mice expressing human tumor necrosis factor alpha (hTNFα). hTNFα mice (C57BL/6 hTNFα) were given a high-fat (23%, w/w) diet containing 20% casein (control group) or 15% FPH and 5% casein (FPH group) for two weeks. After an overnight fast, blood, adipose tissue, and liver samples were collected. Gene expression and enzyme activity was analysed in liver, fatty acid composition was analyzed in liver and ovarian white adipose tissue, and inflammatory parameters, carnitine, and acylcarnitines were analyzed in plasma. The n-3/n-6 fatty acid ratio was higher in mice fed the FPH diet than in mice fed the control diet in both adipose tissue and liver, and the FPH diet affected the gene expression of ∆6 and ∆9 desaturases. Mice fed this diet also demonstrated lower hepatic activity of fatty acid synthase. Concomitantly, a lower plasma INF-γ level was observed. Plasma carnitine and the carnitine precursor γ-butyrobetaine was higher in the FPH-group compared to control, as was plasma short-chained and medium-chained acylcarnitine esters. The higher level of plasma acetylcarnitine may reflect a stimulated mitochondrial and peroxisomal β-oxidation of fatty acids, as the hepatic activities of peroxisomal acyl-CoA oxidase 1 and mitochondrial carnitine palmitoyltransferase-II were higher in the FPH-fed mice. The FPH diet was shown to influence hepatic fatty acid metabolism and fatty acid composition. This indicates that effects on fatty acid metabolism are important for the bioactivity of protein hydrolysates of marine origin.

Bovine glycomacropeptide promotes the growth of Bifidobacterium longum ssp. infantis and modulates its gene expression.

PubMed

O'Riordan, N; O'Callaghan, J; Buttò, L F; Kilcoyne, M; Joshi, L; Hickey, R M

2018-05-23

Bovine milk glycomacropeptide (GMP) is derived from κ-casein, with exclusively o-linked glycosylation. Glycomacropeptide promoted the growth of Bifidobacterium longum ssp. infantis in a concentration-dependent manner, and this activity was lost following periodate treatment of the GMP (GMP-P), which disables biological recognition of the conjugated oligosaccharides. Transcriptional analysis of B. longum ssp. infantis following exposure to GMP revealed a substantial response to GMP relative to bacteria treated with GMP-P, with a greater number of differentially expressed transcripts and larger fold changes versus the control. Therefore, stimulation of B. longum ssp. infantis growth by GMP is intrinsically linked to the peptide's O-linked glycosylation. The pool of differentially expressed transcripts included 2 glycoside hydrolase (family 25) genes, which were substantially upregulated following exposure to GMP, but not GMP-P. These GH25 genes were present in duplicated genomic islands that also contained genes encoding fibronectin type III binding domain proteins and numerous phage-related proteins, all of which were also upregulated. Homologs of this genomic arrangement were present in other Bifidobacterium species, which suggest it may be a conserved domain for the utilization of glycosylated peptides. This study provides insights into the molecular basis for the prebiotic effect of bovine milk GMP on B. longum ssp. infantis. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Dietary soy and meat proteins induce distinct physiological and gene expression changes in rats.

PubMed

Song, Shangxin; Hooiveld, Guido J; Li, Mengjie; Zhao, Fan; Zhang, Wei; Xu, Xinglian; Muller, Michael; Li, Chunbao; Zhou, Guanghong

2016-02-09

This study reports on a comprehensive comparison of the effects of soy and meat proteins given at the recommended level on physiological markers of metabolic syndrome and the hepatic transcriptome. Male rats were fed semi-synthetic diets for 1 wk that differed only regarding protein source, with casein serving as reference. Body weight gain and adipose tissue mass were significantly reduced by soy but not meat proteins. The insulin resistance index was improved by soy, and to a lesser extent by meat proteins. Liver triacylglycerol contents were reduced by both protein sources, which coincided with increased plasma triacylglycerol concentrations. Both soy and meat proteins changed plasma amino acid patterns. The expression of 1571 and 1369 genes were altered by soy and meat proteins respectively. Functional classification revealed that lipid, energy and amino acid metabolic pathways, as well as insulin signaling pathways were regulated differently by soy and meat proteins. Several transcriptional regulators, including NFE2L2, ATF4, Srebf1 and Rictor were identified as potential key upstream regulators. These results suggest that soy and meat proteins induce distinct physiological and gene expression responses in rats and provide novel evidence and suggestions for the health effects of different protein sources in human diets.

Dietary soy and meat proteins induce distinct physiological and gene expression changes in rats

PubMed Central

Song, Shangxin; Hooiveld, Guido J.; Li, Mengjie; Zhao, Fan; Zhang, Wei; Xu, Xinglian; Muller, Michael; Li, Chunbao; Zhou, Guanghong

2016-01-01

This study reports on a comprehensive comparison of the effects of soy and meat proteins given at the recommended level on physiological markers of metabolic syndrome and the hepatic transcriptome. Male rats were fed semi-synthetic diets for 1 wk that differed only regarding protein source, with casein serving as reference. Body weight gain and adipose tissue mass were significantly reduced by soy but not meat proteins. The insulin resistance index was improved by soy, and to a lesser extent by meat proteins. Liver triacylglycerol contents were reduced by both protein sources, which coincided with increased plasma triacylglycerol concentrations. Both soy and meat proteins changed plasma amino acid patterns. The expression of 1571 and 1369 genes were altered by soy and meat proteins respectively. Functional classification revealed that lipid, energy and amino acid metabolic pathways, as well as insulin signaling pathways were regulated differently by soy and meat proteins. Several transcriptional regulators, including NFE2L2, ATF4, Srebf1 and Rictor were identified as potential key upstream regulators. These results suggest that soy and meat proteins induce distinct physiological and gene expression responses in rats and provide novel evidence and suggestions for the health effects of different protein sources in human diets. PMID:26857845

Lower weight gain and hepatic lipid content in hamsters fed high fat diets supplemented with white rice protein, brown rice protein, soy protein, and their hydrolysates.

PubMed

Zhang, Huijuan; Bartley, Glenn E; Mitchell, Cheryl R; Zhang, Hui; Yokoyama, Wallace

2011-10-26

The physiological effects of the hydrolysates of white rice protein (WRP), brown rice protein (BRP), and soy protein (SP) hydrolyzed by the food grade enzyme, alcalase2.4 L, were compared to the original protein source. Male Syrian Golden hamsters were fed high-fat diets containing either 20% casein (control) or 20% extracted proteins or their hydrolysates as the protein source for 3 weeks. The brown rice protein hydrolysate (BRPH) diet group reduced weight gain 76% compared with the control. Animals fed the BRPH supplemented diet also had lower final body weight, liver weight, very low density lipoprotein cholesterol (VLDL-C), and liver cholesterol, and higher fecal fat and bile acid excretion than the control. Expression levels of hepatic genes for lipid oxidation, PPARα, ACOX1, and CPT1, were highest for hamsters fed the BRPH supplemented diet. Expression of CYP7A1, the gene regulating bile acid synthesis, was higher in all test groups. Expression of CYP51, a gene coding for an enzyme involved in cholesterol synthesis, was highest in the BRPH diet group. The results suggest that BRPH includes unique peptides that reduce weight gain and hepatic cholesterol synthesis.

CKB1 is involved in abscisic acid and gibberellic acid signaling to regulate stress responses in Arabidopsis thaliana.

PubMed

Yuan, Congying; Ai, Jianping; Chang, Hongping; Xiao, Wenjun; Liu, Lu; Zhang, Cheng; He, Zhuang; Huang, Ji; Li, Jinyan; Guo, Xinhong

2017-05-01

Casein kinase II (CK2), an evolutionarily well-conserved Ser/Thr kinase, plays critical roles in all higher organisms including plants. CKB1 is a regulatory subunit beta of CK2. In this study, homozygous T-DNA mutants (ckb1-1 and ckb1-2) and over-expression plants (35S:CKB1-1, 35S:CKB1-2) of Arabidopsis thaliana were studied to understand the role of CKB1 in abiotic stress and gibberellic acid (GA) signaling. Histochemical staining showed that although CKB1 was expressed in all organs, it had a relatively higher expression in conducting tissues. The ckb1 mutants showed reduced sensitivity to abscisic acid (ABA) during seed germination and seedling growth. The increased stomatal aperture, leaf water loss and proline accumulation were observed in ckb1 mutants. In contrast, the ckb1 mutant had increased sensitivity to polyaluminum chloride during seed germination and hypocotyl elongation. We obtained opposite results in over-expression plants. The expression levels of a number of genes in the ABA and GA regulatory network had changed. This study demonstrates that CKB1 is an ABA signaling-related gene, which subsequently influences GA metabolism, and may play a positive role in ABA signaling.

Reproductive Experience Alters Prolactin Receptor Expression in Mammary and Hepatic Tissues in Female Rats1

PubMed Central

Bridges, Robert S.; Scanlan, Victoria F.; Lee, Jong-O; Byrnes, Elizabeth M.

2011-01-01

Recent studies have reported that reproductive experience in female rats alters prolactin (PRL) receptor gene expression in the brain as well as neural sensitivity to PRL. Given PRL's actions in nonneural tissues, that is, mammary tissue and liver, it was asked whether reproductive experience may also alter prolactin receptor (Prlr) gene expression in these tissues. Groups of age-matched female rats were generated with varying reproductive histories. Separate groups of primiparous (first lactation) and multiparous (second lactation) had mammary tissue and liver samples collected on Day 3 or 10 of lactation. A fifth group raised one litter to weaning and then resumed estrous cyclicity. This group and a final group of age-matched, virgin controls were killed on diestrus. Tissue was processed by quantitative PCR for expression rates of the long and short forms of Prlr mRNA as well as casein beta mRNA (mammary tissue only). Western blots were performed to quantify receptor protein content. Multiple lactations as well as lactation itself resulted in alterations in Prlr expression. Prlr gene expression in mammary tissue was increased in primiparous mothers compared with that in multiparous dams, whereas in the liver, Prlr expression was reduced during an initial lactation. In contrast, PRLR protein levels declined during lactation in mammary, but not hepatic, tissues. Overall, the results demonstrate that the prolactin receptor system is altered in nonneural tissues as a result of the female's reproductive history. The findings are discussed in the context of milk and bile production and PRL's possible role in breast cancer. PMID:21508351

Reproductive experience alters prolactin receptor expression in mammary and hepatic tissues in female rats.

PubMed

Bridges, Robert S; Scanlan, Victoria F; Lee, Jong-O; Byrnes, Elizabeth M

2011-08-01

Recent studies have reported that reproductive experience in female rats alters prolactin (PRL) receptor gene expression in the brain as well as neural sensitivity to PRL. Given PRL's actions in nonneural tissues, that is, mammary tissue and liver, it was asked whether reproductive experience may also alter prolactin receptor (Prlr) gene expression in these tissues. Groups of age-matched female rats were generated with varying reproductive histories. Separate groups of primiparous (first lactation) and multiparous (second lactation) had mammary tissue and liver samples collected on Day 3 or 10 of lactation. A fifth group raised one litter to weaning and then resumed estrous cyclicity. This group and a final group of age-matched, virgin controls were killed on diestrus. Tissue was processed by quantitative PCR for expression rates of the long and short forms of Prlr mRNA as well as casein beta mRNA (mammary tissue only). Western blots were performed to quantify receptor protein content. Multiple lactations as well as lactation itself resulted in alterations in Prlr expression. Prlr gene expression in mammary tissue was increased in primiparous mothers compared with that in multiparous dams, whereas in the liver, Prlr expression was reduced during an initial lactation. In contrast, PRLR protein levels declined during lactation in mammary, but not hepatic, tissues. Overall, the results demonstrate that the prolactin receptor system is altered in nonneural tissues as a result of the female's reproductive history. The findings are discussed in the context of milk and bile production and PRL's possible role in breast cancer.

Effects of glutamine supplementation on oxidative stress-related gene expression and antioxidant properties in rats with streptozotocin-induced type 2 diabetes.

PubMed

Tsai, Pei-Hsuan; Liu, Jun-Jen; Yeh, Chui-Li; Chiu, Wan-Chun; Yeh, Sung-Ling

2012-04-01

There are close links among hyperglycaemia, oxidative stress and diabetic complications. Glutamine (GLN) is an amino acid with immunomodulatory properties. The present study investigated the effect of dietary GLN on oxidative stress-relative gene expressions and tissue oxidative damage in diabetes. There were one normal control (NC) and two diabetic groups in the present study. Diabetes was induced by an intraperitoneal injection of nicotinamide followed by streptozotocin (STZ). Rats in the NC group were fed a regular chow diet. In the two diabetic groups, one group (diabetes mellitus, DM) was fed a common semi-purified diet while the other group received a diet in which part of the casein was replaced by GLN (DM-GLN). GLN provided 25% of total amino acid N. The experimental groups were fed the respective diets for 8 weeks, and then the rats were killed for further analysis. The results showed that blood thioredoxin-interacting protein (Txnip) mRNA expression in the diabetic groups was higher than that in the NC group. Compared with the DM group, the DM-GLN group had lower glutamine fructose-6-phosphate transaminase 1, a receptor of advanced glycation end products, and Txnip gene expressions in blood mononuclear cells. The total antioxidant capacity was lower and antioxidant enzyme activities were altered by the diabetic condition. GLN supplementation increased antioxidant capacity and normalised antioxidant enzyme activities. Also, the renal nitrotyrosine level and Txnip mRNA expression were lower when GLN was administered. These results suggest that dietary GLN supplementation decreases oxidative stress-related gene expression, increases the antioxidant potential and may consequently attenuate renal oxidative damage in rats with STZ-induced diabetes.

Protective effect of soybeans as protein source in the diet against cadmium-aorta redox and morphological alteration

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pérez Díaz, Matías F.F.; Acosta, Mariano; Mohamed, Fabián H.

We investigated the effects of cadmium exposition on thoracic aorta redox status and morphology, and the putative protective effect of soybeans in the diet. Male Wistar rats were separated into 6 groups: 3 fed with a diet containing casein and 3 containing soybeans, as protein source. Within each protein group, one was given tap water (control) and the other two tap water containing 15 and 100 ppm of Cd{sup 2+}, respectively, for two months. In rats fed with casein diet, 15 ppm of Cd induced an increase of thiobarbituric acid-reactive substances (TBARS), and of the catalase (CAT) and glutathione peroxidasemore » (GPx) activities, which were even higher with 100 ppm of Cd{sup 2+}, in aorta. Also, 100 ppm Cd{sup 2+} exposure increased superoxide dismutase (CuZnSOD) activity; CAT, GPX, SOD, Nrf2 and metallothioneine II mRNA expressions and CAT, GPx and NOX-2 protein levels, compared with control. Aorta endothelial and cytoplasmic alterations were observed. However, with the soybeans diet, 15 and 100 ppm of Cd{sup 2+} did not modify TBARS levels; CAT, GPX and Nrf2 mRNA expressions; CAT, GPx and NOX-2 protein; and the aorta morphology, compared with control. The soybean diet attenuates the redox changes and protects against morphological alterations induced, in a dose-dependent way, by Cd in aorta. - Highlights: • Under casein diet, 100 ppm Cd{sup 2+} in drinking water induces oxidative stress in aorta. • Under casein diet, 100 ppm Cd{sup 2+} increases Nrf2, MT II and NOX2 expressions in aorta. • Under casein diet, 100 ppm Cd{sup 2+} induces morphological changes in rat aorta. • The soybean diet attenuates the redox changes induced by Cd in rat aorta. • The soybean diet attenuates morphological alterations induced by Cd in rat aorta.« less

Casein kinase 2 (CK2) increases survivin expression via enhanced β-catenin–T cell factor/lymphoid enhancer binding factor-dependent transcription

PubMed Central

Tapia, J. C.; Torres, V. A.; Rodriguez, D. A.; Leyton, L.; Quest, A. F. G.

2006-01-01

Increased expression of casein kinase 2 (CK2) is associated with hyperproliferation and suppression of apoptosis in cancer. Mutations in the tumor suppressor APC (adenomatous polyposis coli) are frequent in colon cancer and often augment β-catenin–T cell factor (Tcf)/lymphoid enhancer binding factor (Lef)-dependent transcription of genes such as c-myc and cyclin-D1. CK2 has also been implicated recently in the regulation of β-catenin stability. To identify mechanisms by which CK2 promotes survival, effects of the specific CK2 inhibitors 4,5,6,7-tetrabromobenzotriazole (TBB) and 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole were assessed. TBB and 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole significantly decreased proliferation and increased apoptosis of HT29(US) colon cancer cells. RT-PCR and immunoblot analysis revealed that both inhibitors decreased survivin mRNA and protein levels in HT29(US) cells. Similar effects were observed with TBB in human DLD-1 and SW-480 colorectal cells as well as ZR-75 breast cancer cells and HEK-293T embryonic kidney cells. Expression of GFP–CK2α in HEK-293T cells resulted in β-catenin–Tcf/Lef-dependent up-regulation of survivin and increased resistance to anticancer drugs. Augmented β-catenin–Tcf/Lef-dependent transcription and resistance to apoptosis observed upon GFP–CK2α expression were abolished by TBB. Alternatively, HEK-293T cells expressing GFP–survivin were resistant to TBB-induced apoptosis. Finally, siRNA-mediated down-regulation of CK2α in HEK-293T cells coincided with reduced β-catenin and survivin levels. Taken together, these results suggest that CK2 kinase activity promotes survival by increasing survivin expression via β-catenin–Tcf/Lef-mediated transcription. Hence, selective CK2 inhibition or down-regulation in tumors may provide an attractive opportunity for the development of novel cancer therapies. PMID:17005722

Molecular characterization of a long range haplotype affecting protein yield and mastitis susceptibility in Norwegian Red cattle.

PubMed

Sodeland, Marte; Grove, Harald; Kent, Matthew; Taylor, Simon; Svendsen, Morten; Hayes, Ben J; Lien, Sigbjørn

2011-08-11

Previous fine mapping studies in Norwegian Red cattle (NRC) in the region 86-90.4 Mb on Bos taurus chromosome 6 (BTA6) has revealed a quantitative trait locus (QTL) for protein yield (PY) around 88 Mb and a QTL for clinical mastitis (CM) around 90 Mb. The close proximity of these QTLs may partly explain the unfavorable genetic correlation between these two traits in NRC. A long range haplotype covering this region was introduced into the NRC population through the importation of a Holstein-Friesian bull (1606 Frasse) from Sweden in the 1970s. It has been suggested that this haplotype has a favorable effect on milk protein content but an unfavorable effect on mastitis susceptibility. Selective breeding for milk production traits is likely to have increased the frequency of this haplotype in the NRC population. Association mapping for PY and CM in NRC was performed using genotypes from 556 SNPs throughout the region 86-97 Mb on BTA6 and daughter-yield-deviations (DYDs) from 2601 bulls made available from the Norwegian dairy herd recording system. Highest test scores for PY were found for single-nucleotide polymorphisms (SNPs) within and surrounding the genes CSN2 and CSN1S2, coding for the β-casein and α(S2)-casein proteins. High coverage re-sequencing by high throughput sequencing technology enabled molecular characterization of a long range haplotype from 1606 Frasse encompassing these two genes. Haplotype analysis of a large number of descendants from this bull indicated that the haplotype was not markedly disrupted by recombination in this region. The haplotype was associated with both increased milk protein content and increased susceptibility to mastitis, which might explain parts of the observed genetic correlation between PY and CM in NRC. Plausible causal polymorphisms affecting PY were detected in the promoter region and in the 5'-flanking UTR of CSN1S2. These polymorphisms could affect transcription or translation of CSN1S2 and thereby affect the amount of α(S2)-casein in milk. Highest test scores for CM were found in the region 89-91 Mb on BTA6, very close to a cluster of genes coding for CXC chemokines. Expression levels of some of these CXC chemokines have previously been shown to increase in bovine mammary gland cell lines after exposure to bacterial cell wall components. Molecular characterization of the long range haplotype from the Holstein-Friesian bull 1606 Frasse, imported into NRC in the 1970s, revealed polymorphisms that could affect transcription or translation of the casein gene CSN1S2. Sires with this haplotype had daughters with significantly elevated milk protein content and selection for milk production traits is likely to have increased the frequency of this haplotype in the NRC population. The haplotype was also associated with increased mastitis susceptibility, which might explain parts of the genetic correlation between PY and CM in NRC.

Lipolysis and proteolysis profiles of fresh artisanal goat cheese made with raw milk with 3 different fat contents.

PubMed

Sánchez-Macías, D; Morales-Delanuez, A; Moreno-Indias, I; Hernández-Castellano, L E; Mendoza-Grimón, V; Castro, N; Argüello, A

2011-12-01

The objective of this study was to describe the proteolysis and lipolysis profiles in goat cheese made in the Canary Islands (Spain) using raw milk with 3 different fat contents (0.5, 1.5, and 5%) and ripened for 1, 7, 14, and 28 d. β-Casein was the most abundant protein in all cheeses and at all ripening times. Quantitative analysis showed a general decrease in caseins as ripening progressed, and degradation rates were higher for α(S1)-casein than for β-casein and α(S2)-casein. Furthermore, the degradation rate during the experimental time decreased with lower fat contents. The α(S2)-casein and α(S1)-casein levels that remained in full-fat and reduced-fat cheeses were less than those in low-fat cheese. In contrast, β-casein also showed degradation along with ripening, but differences in degradation among the 3 cheese types were not significant at 28 d. The degradation products increased with the ripening time in all cheeses, but they were higher in full-fat cheese than in reduced-fat and low-fat cheeses. The free fatty acid concentration per 100g of cheese was higher in full-fat cheese than in reduced- and low-fat cheese; however, when the results were expressed as milligrams of free fatty acids per gram of fat in cheese, then lipolysis occurred more rapidly in low-fat cheese than in reduced- and full-fat cheeses. These results may explain the atypical texture and off-flavors found in low-fat goat cheeses, likely the main causes of non-acceptance. Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Influence of micellar calcium and phosphorus on rennet coagulation properties of cows milk.

PubMed

Malacarne, Massimo; Franceschi, Piero; Formaggioni, Paolo; Sandri, Sandro; Mariani, Primo; Summer, Andrea

2014-05-01

The main requirement for milk processed in most cheese typologies is its rennet coagulation ability. Despite the increasing number of studies, the causes for abnormal coagulation of milk are not fully understood. The aim of this study was to ascertain relationships between milk characteristics and its rennet coagulation ability, focusing on the influence of calcium (Ca) and phosphorus (P). Ca and P are essential constituents of the micelles. Micellar P can be present as part of colloidal calcium phosphate (inorganic-P) or covalently bound to caseins as phosphate groups (casein-P). Eighty one herd milk samples (SCC<400 000 cell/ml) were classified as Optimal (8), Suboptimal (39) Poor (29) and Non-coagulating milk (5), according to their rennet coagulation parameters as assessed by lactodynamographic test. Samples were analysed for their chemical composition (basic composition, protein fractions, minerals and salt equilibria), physicochemical parameters (pH and titratable acidity) and rheological properties. Optimal milk was characterised by the highest contents of major constituents, protein fractions and minerals, lowest content of chloride and highest values of titratable acidity. Non-coagulating milk was characterised by the highest values of pH and the lowest of titratable acidity. At micellar level, Optimal milk showed the highest values of colloidal Ca, casein-P and colloidal Mg (g/100 g casein), while Non-coagulating milk showed the lowest values. Interestingly, there was no statistical difference regarding the content of colloidal inorganic-P (g/100 g casein) between Optimal and Non-coagulating milks. Overall, high mineralisation of the micelle (expressed as g inorganic-P/100 g casein) positively affect its rennetability. However, excessive mineralisation could lead to a reduction of the phosphate groups (g casein-P/100 g casein) available for curd formation.

Molecular cloning, overexpression, purification, and sequence analysis of the giant panda (Ailuropoda melanoleuca) ferritin light polypeptide.

PubMed

Fu, L; Hou, Y L; Ding, X; Du, Y J; Zhu, H Q; Zhang, N; Hou, W R

2016-08-30

The complementary DNA (cDNA) of the giant panda (Ailuropoda melanoleuca) ferritin light polypeptide (FTL) gene was successfully cloned using reverse transcription-polymerase chain reaction technology. We constructed a recombinant expression vector containing FTL cDNA and overexpressed it in Escherichia coli using pET28a plasmids. The expressed protein was then purified by nickel chelate affinity chromatography. The cloned cDNA fragment was 580 bp long and contained an open reading frame of 525 bp. The deduced protein sequence was composed of 175 amino acids and had an estimated molecular weight of 19.90 kDa, with an isoelectric point of 5.53. Topology prediction revealed one N-glycosylation site, two casein kinase II phosphorylation sites, one N-myristoylation site, two protein kinase C phosphorylation sites, and one cell attachment sequence. Alignment indicated that the nucleotide and deduced amino acid sequences are highly conserved across several mammals, including Homo sapiens, Cavia porcellus, Equus caballus, and Felis catus, among others. The FTL gene was readily expressed in E. coli, which gave rise to the accumulation of a polypeptide of the expected size (25.50 kDa, including an N-terminal polyhistidine tag).

Impairment of male reproductive function after sleep deprivation.

PubMed

Alvarenga, Tathiana A; Hirotsu, Camila; Mazaro-Costa, Renata; Tufik, Sergio; Andersen, Monica L

2015-05-01

To evaluate the influence of sleep loss on sexual behavior, hormone levels, sperm parameters, and testis-specific gene expression in male rats. Experimental research. Animal laboratory. Male adult Wistar-Hannover rats. Sexually experienced rats were subjected to paradoxic sleep deprivation (PSD) for 96 hours or sleep restriction (SR) for 21 days or kept in their home cage as control (CTRL). Sexual behavior, hormone levels, sperm parameters and expression of stress and nitric oxide-related genes were evaluated. PSD significantly decreased sexual behavior compared with the CTRL group, whereas SR had no effect. The PSD group had significantly lower testosterone levels than the CTRL group. Both PSD and SR groups had lower sperm viabilities than the CTRL group. The decrease in the number of live sperm compared with the CTRL group was larger in the PSD group than in the SR group. Regarding testicular gene expression, both PSD and SR led to an increase of iNOS and hydroxysteroid 11β-dehydrogenase 1 expressions compared with the CTRL group. These changes were more pronounced in the PSD group. A significant increase in endothelial nitric oxide synthase expression was observed in the PSD groups compared with the CTRL group. No changes were observed in dimethylarginine dimethylaminohydrolase 1 and casein kinase 2β-polypeptide expressions. Sleep loss can promote marked changes in the male reproductive system of rats, particularly affecting spermatic function in part by interfering in the testicular nitric oxide pathway. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Translation attenuation via 3′ terminal codon usage in bovine csn1s2 is responsible for the difference in αs2- and β-casein profile in milk

PubMed Central

Kim, Julie J; Yu, Jaeju; Bag, Jnanankur; Bakovic, Marica; Cant, John P

2015-01-01

The rate of secretion of αs2-casein into bovine milk is approximately 25% of that of β-casein, yet mammary expression of their respective mRNA transcripts (csn1s2 and csn2) is not different. Our objective was to identify molecular mechanisms that explain the difference in translation efficiency between csn1s2 and csn2. Cell-free translational efficiency of csn2 was 5 times that of csn1s2. Transcripts of csn1s2 distributed into heavier polysomes than csn2 transcripts, indicating an attenuation of elongation and/or termination. Stimulatory and inhibitory effects of the 5′ and 3′ UTRs on translational efficiency were different with luciferase and casein sequences in the coding regions. Substituting the 5′ and 3′ UTRs from csn2 into csn1s2 did not improve csn1s2 translation, implicating the coding region itself in the translation difference. Deletion of a 28-codon fragment from the 3′ terminus of the csn1s2 coding region, which displays codons with low correlations to cell fitness, increased translation to a par with csn2. We conclude that the usage of the last 28 codons of csn1s2 is the main regulatory element that attenuates its expression and is responsible for the differential translational expression of csn1s2 and csn2. PMID:25826667

Short communication: casein haplotype variability in sicilian dairy goat breeds.

PubMed

Gigli, I; Maizon, D O; Riggio, V; Sardina, M T; Portolano, B

2008-09-01

In the Mediterranean region, goat milk production is an important economic activity. In the present study, 4 casein genes were genotyped in 5 Sicilian goat breeds to 1) identify casein haplotypes present in the Argentata dell'Etna, Girgentana, Messinese, Derivata di Siria, and Maltese goat breeds; and 2) describe the structure of the Sicilian goat breeds based on casein haplotypes and allele frequencies. In a sample of 540 dairy goats, 67 different haplotypes with frequency >or=0.01 and 27 with frequency >or=0.03 were observed. The most common CSN1S1-CSN2-CSN1S2-CSN3 haplotype for Derivata di Siria and Maltese was FCFB (0.17 and 0.22, respectively), whereas for Argentata dell'Etna, Girgentana and Messinese was ACAB (0.06, 0.23, and 0.10, respectively). According to the haplotype reconstruction, Argentata dell'Etna, Girgentana, and Messinese breeds presented the most favorable haplotype for cheese production, because the casein concentration in milk of these breeds might be greater than that in Derivata di Siria and Maltese breeds. Based on a cluster analysis, the breeds formed 2 main groups: Derivata di Siria, and Maltese in one group, and Argentata dell'Etna and Messinese in the other; the Girgentana breed was between these groups but closer to the latter.

Divergence at the casein haplotypes in dairy and meat goat breeds.

PubMed

Küpper, Julia; Chessa, Stefania; Rignanese, Daniela; Caroli, Anna; Erhardt, Georg

2010-02-01

Casein genes have been proved to have an influence on milk properties, and are in addition appropriate for phylogeny studies. A large number of casein polymorphisms exist in goats, making their analysis quite complex. The four casein loci were analyzed by molecular techniques for genetic polymorphism detection in the two dairy goat breeds Bunte Deutsche Edelziege (BDE; n=96), Weisse Deutsche Edelziege (WDE; n=91), and the meat goat breed Buren (n=75). Of the 35 analyzed alleles, 18 were found in BDE, and 17 in Buren goats and WDE. In addition, a new allele was identified at the CSN1S1 locus in the BDE, showing a frequency of 0.05. This variant, named CSN1S1*A', is characterized by a t-->c transversion in intron 9. Linkage disequilibrium was found at the casein haplotype in all three breeds. A total of 30 haplotypes showed frequencies higher than 0.01. In the Buren breed only one haplotype showed a frequency higher than 0.1. The ancestral haplotype B-A-A-B (in the order: CSN1S1-CSN2-CSN1S2-CSN3) occurred in all three breeds, showing a very high frequency (>0.8) in the Buren.

Use of the yeast-like cells of Tremella fuciformis as a cell factory to produce a Pleurotus ostreatus hydrophobin.

PubMed

Zhu, Hanyu; Liu, Dongmei; Wang, Yuanyuan; Ren, Danfeng; Zheng, Liesheng; Chen, Liguo; Ma, Aimin

2017-08-01

To obtain hydrophobin, a Class I hydrophobin gene, Po.hyd from Pleurotus ostreatus, was transformed into the yeast-like cells of Tremella fuciformis using Agrobacterium tumefaciens. The hydrophobin Po.HYD from P. ostreatus was heterogeneously expressed by the yeast-like cells of T. fuciformis. Plasmids harboring the Po.hyd gene driven by endogenous glyceraldehyde-3-phosphate dehydrogenase promoter were transformed by A. tumefaciens. The integration and expression of the rPo.HYD in the T. fuciformis cells were confirmed by PCR, Southern blot, fluorescence microscopy and quantitative real-time PCR. SDS-PAGE demonstrated that the rPo.HYD was extracted with the expected MW of 14 kDa. The yield of purified rPo.HYD was 0.58 mg/g dry wt. The protein, with its ability to stabilize oil droplets, exhibited a better emulsifying activity than the typical food emulsifiers Tween 20 and sodium caseinate. Tremella fuciformis can be used as a cell factory to produce hydrophobin on a large scale for the food industry.

Differential Response of Extracellular Proteases of Trichoderma Harzianum Against Fungal Phytopathogens.

PubMed

Sharma, Vivek; Salwan, Richa; Sharma, Prem N

2016-09-01

In the present study, production of extracellular proteases by Trichoderma harzianum was evaluated based on the relative gene expression and spectrophotometric assay. The fungal isolates were grown in Czapek Dox Broth medium supplemented with deactivated mycelium of plant fungal pathogens such as Fusarium oxysporum, Colletotrichum capsici, Gloeocercospora sorghi, and Colletotrichum truncatum. The maximum protease activity was detected after 48 h of incubation against Colletotrichum spp. Similarly in qRT-PCR, the relative gene expression of four proteases varied from 48 to 96 h against host pathogens in a time-independent manner. Among proteases, statistically significant upregulation of asp, asp, and srp was observed against Colletotrichum spp., followed by F. oxysporum. But in the case of pepM22, maximum upregulation was observed against F. oxysporum. The variation in enzyme assay and qRT-PCR of proteases at different time intervals against various fungal phytopathogens could be due to the limitation of using casein as a substrate for all types of proteases or protease-encoding transcripts selected for qRT-PCR, which may not be true representative of total protease activity.

A tree of life based on ninety-eight expressed genes conserved across diverse eukaryotic species

PubMed Central

Jayaswal, Pawan Kumar; Dogra, Vivek; Shanker, Asheesh; Sharma, Tilak Raj

2017-01-01

Rapid advances in DNA sequencing technologies have resulted in the accumulation of large data sets in the public domain, facilitating comparative studies to provide novel insights into the evolution of life. Phylogenetic studies across the eukaryotic taxa have been reported but on the basis of a limited number of genes. Here we present a genome-wide analysis across different plant, fungal, protist, and animal species, with reference to the 36,002 expressed genes of the rice genome. Our analysis revealed 9831 genes unique to rice and 98 genes conserved across all 49 eukaryotic species analysed. The 98 genes conserved across diverse eukaryotes mostly exhibited binding and catalytic activities and shared common sequence motifs; and hence appeared to have a common origin. The 98 conserved genes belonged to 22 functional gene families including 26S protease, actin, ADP–ribosylation factor, ATP synthase, casein kinase, DEAD-box protein, DnaK, elongation factor 2, glyceraldehyde 3-phosphate, phosphatase 2A, ras-related protein, Ser/Thr protein phosphatase family protein, tubulin, ubiquitin and others. The consensus Bayesian eukaryotic tree of life developed in this study demonstrated widely separated clades of plants, fungi, and animals. Musa acuminata provided an evolutionary link between monocotyledons and dicotyledons, and Salpingoeca rosetta provided an evolutionary link between fungi and animals, which indicating that protozoan species are close relatives of fungi and animals. The divergence times for 1176 species pairs were estimated accurately by integrating fossil information with synonymous substitution rates in the comprehensive set of 98 genes. The present study provides valuable insight into the evolution of eukaryotes. PMID:28922368

Soya protein attenuates abnormalities of the renin-angiotensin system in adipose tissue from obese rats.

PubMed

Frigolet, María E; Torres, Nimbe; Tovar, Armando R

2012-01-01

Several metabolic disturbances during obesity are associated with adipose tissue-altered functions. Adipocytes contain the renin-angiotensin system (RAS), which regulates signalling pathways that control angiogenesis via Akt in an autocrine fashion. Soya protein (Soy) consumption modifies the gene expression pattern in adipose tissue, resulting in an improved adipocyte function. Therefore, the aim of the present work is to study whether dietary Soy regulates the expression of RAS and angiogenesis-related genes and its association with the phosphorylated state of Akt in the adipose tissue of obese rats. Animals were fed a 30 % Soy or casein (Cas) diet containing 5 or 25 % fat for 160 d. mRNA abundance was studied in the adipose tissue, and Akt phosphorylation and hormone release were measured in the primary adipocyte culture. The present results show that Soy treatment in comparison with Cas consumption induces lower angiotensin release and increased insulin-stimulated Akt activation in adipocytes. Furthermore, Soy consumption varies the expression of RAS and angiogenesis-related genes, which maintain cell size and vascularity in the adipose tissue of rats fed a high-fat diet. Thus, adipocyte hypertrophy and impaired angiogenesis, which are frequently observed in dysfunctional adipose tissue, were avoided by consuming dietary Soy. Taken together, these findings suggest that Soy can be used as a dietary strategy to preserve adipocyte functionality and to prevent obesity abnormalities.

Targeted microRNA expression in dairy cattle directs production of β-lactoglobulin-free, high-casein milk

PubMed Central

Jabed, Anower; Wagner, Stefan; McCracken, Judi; Wells, David N.; Laible, Goetz

2012-01-01

Milk from dairy cows contains the protein β-lactoglobulin (BLG), which is not present in human milk. As it is a major milk allergen, we wished to decrease BLG levels in milk by RNAi. In vitro screening of 10 microRNAs (miRNAs), either individually or in tandem combinations, identified several that achieved as much as a 98% knockdown of BLG. One tandem construct was expressed in the mammary gland of an ovine BLG-expressing mouse model, resulting in 96% knockdown of ovine BLG in milk. Following this in vivo validation, we produced a transgenic calf, engineered to express these tandem miRNAs. Analysis of hormonally induced milk from this calf demonstrated absence of BLG and a concurrent increase of all casein milk proteins. The findings demonstrate miRNA–mediated depletion of an allergenic milk protein in cattle and validate targeted miRNA expression as an effective strategy to alter milk composition and other livestock traits. PMID:23027958

Two distinct phases of apoptosis in mammary gland involution: proteinase-independent and -dependent pathways

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lund, Leif R; Romer, John; Thomasset, Nicole

Postlactational involution of the mammary gland is characterized by two distinct physiological events: apoptosis of the secretory, epithelial cells undergoing programmed cell death, and proteolytic degradation of the mammary gland basement membrane. We examined the spatial and temporal patterns of apoptotic cells in relation to those of proteinases during involution of the BALB/c mouse mammary gland. Apoptosis was almost absent during lactation but became evident at day 2 of involution, when {beta}-casein gene expression was still high. Apoptotic cells were then seen at least up to day 8 of involution, when {beta}-casein gene expression was being extinguished. Expression of sulfatedmore » glycoprotein-2 (SGP-2), interleukin-1{beta} converting enzyme (ICE) and tissue inhibitor of metalloproteinases-1 was upregulated at day 2, when apoptotic cells were seen initially. Expression of the matrix metalloproteinases gelatinase A and stromelysin-1 and the serine proteinase urokinase-type plasminogen activator, which was low during lactation, was strongly upregulated in parallel starting at day 4 after weaning, coinciding with start of the collapse of the lobulo-alveolar structures and the intensive tissue remodeling in involution. The major sites of mRNA synthesis for these proteinases were fibroblast-like cells in the periductal stroma and stromal cells surrounding the collapsed alveoli, suggesting that the degradative phase of involution is due to a specialized mesenchymal-epithelial interaction. To elucidate the functional role of these proteinases during involution, at the onset of weaning we treated mice systemically with the glucocorticoid hydrocortisone, which is known to inhibit mammary gland involution. Although the initial wave of apoptotic cells appeared in the lumina of the gland, the dramatic regression and tissue remodeling usually evident by day 5 was substantially inhibited by systemic treatment with hydrocortisone. mRNA and protein for gelatinase A, stromelysin-1 and uPA were weakly induced, if at all, in hydrocortisonetreated mice. Furthermore, mRNA for membrane-type matrix metalloproteinase decreased after hydrocortisone treatment and paralleled the almost complete inhibition of activation of latent gelatinase A. Concomitantly, the gland filled with an overabundance of milk. Our data support the hypothesis that there are at least two distinct phases of involution: an initial phase, characterized by induction of the apoptosis-associated genes SGP-2 and ICE and apoptosis of fully differentiated mammary epithelial cells without visible degradation of the extracellular matrix, and a second phase, characterized by extracellular matrix remodeling and altered mesenchymal-epithelial interactions, followed by apoptosis of cells that are losing differentiated functions.« less

Longitudinal data analysis of polymorphisms in the κ-casein and β-lactoglobulin genes shows differential effects along the trajectory of the lactation curve in tropical dairy goats.

PubMed

Cardona, Samir Julián Calvo; Cadavid, Henry Cardona; Corrales, Juan David; Munilla, Sebastián; Cantet, Rodolfo J C; Rogberg-Muñoz, Andrés

2016-09-01

The κ-casein (CSN-3) and β-lactoglobulin (BLG) genes are extensively polymorphic in ruminants. Several association studies have estimated the effects of polymorphisms in these genes on milk yield, milk composition, and cheese-manufacturing properties. Usually, these results are based on production integrated over the lactation curve or on cross-sectional studies at specific days in milk (DIM). However, as differential expression of milk protein genes occurs over lactation, the effect of the polymorphisms may change over time. In this study, we fitted a mixed-effects regression model to test-day records of milk yield and milk quality traits (fat, protein, and total solids yields) from Colombian tropical dairy goats. We used the well-characterized A/B polymorphisms in the CSN-3 and BLG genes. We argued that this approach provided more efficient estimators than cross-sectional designs, given the same number and pattern of observations, and allowed exclusion of between-subject variation from model error. The BLG genotype AA showed a greater performance than the BB genotype for all traits along the whole lactation curve, whereas the heterozygote showed an intermediate performance. We observed no such constant pattern for the CSN-3 gene between the AA homozygote and the heterozygote (the BB genotype was absent from the sample). The differences among the genotypic effects of the BLG and the CSN-3 polymorphisms were statistically significant during peak and mid lactation (around 40-160 DIM) for the BLG gene and only for mid lactation (80-145 DIM) for the CSN-3 gene. We also estimated the additive and dominant effects of the BLG locus. The locus showed a statistically significant additive behavior along the whole lactation trajectory for all quality traits, whereas for milk yield the effect was not significant at later stages. In turn, we detected a statistically significant dominance effect only for fat yield in the early and peak stages of lactation (at about 1-45 DIM). The longitudinal analysis of test-day records allowed us to estimate the differential effects of polymorphisms along the lactation curve, pointing toward stages that could be affected by the gene. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Fermentation of soy milk via Lactobacillus plantarum improves dysregulated lipid metabolism in rats on a high cholesterol diet.

PubMed

Kim, Yunhye; Yoon, Sun; Lee, Sun Bok; Han, Hye Won; Oh, Hayoun; Lee, Wu Joo; Lee, Seung-Min

2014-01-01

We aimed to investigate whether in vitro fermentation of soy with L. plantarum could promote its beneficial effects on lipids at the molecular and physiological levels. Rats were fed an AIN76A diet containing 50% sucrose (w/w) (CTRL), a modified AIN76A diet supplemented with 1% (w/w) cholesterol (CHOL), or a CHOL diet where 20% casein was replaced with soy milk (SOY) or fermented soy milk (FSOY). Dietary isoflavone profiles, serum lipids, hepatic and fecal cholesterol, and tissue gene expression were examined. The FSOY diet had more aglycones than did the SOY diet. Both the SOY and FSOY groups had lower hepatic cholesterol and serum triglyceride (TG) than did the CHOL group. Only FSOY reduced hepatic TG and serum free fatty acids and increased serum HDL-CHOL and fecal cholesterol. Compared to CHOL, FSOY lowered levels of the nuclear forms of SREBP-1c and SREBP-2 and expression of their target genes, including FAS, SCD1, LDLR, and HMGCR. On the other hand, FSOY elevated adipose expression levels of genes involved in TG-rich lipoprotein uptake (ApoE, VLDLR, and Lrp1), fatty acid oxidation (PPARα, CPT1α, LCAD, CYP4A1, UCP2, and UCP3), HDL-biogenesis (ABCA1, ApoA1, and LXRα), and adiponectin signaling (AdipoQ, AdipoR1, and AdipoR2), as well as levels of phosphorylated AMPK and ACC. SOY conferred a similar expression profile in both liver and adipose tissues but failed to reach statistical significance in many of the genes tested, unlike FSOY. Our data indicate that fermentation may be a way to enhance the beneficial effects of soy on lipid metabolism, in part via promoting a reduction of SREBP-dependent cholesterol and TG synthesis in the liver, and enhancing adiponectin signaling and PPARα-induced expression of genes involved in TG-rich lipoprotein clearance, fatty acid oxidation, and reverse cholesterol transport in adipose tissues.

Fermentation of Soy Milk via Lactobacillus plantarum Improves Dysregulated Lipid Metabolism in Rats on a High Cholesterol Diet

PubMed Central

Han, Hye Won; Oh, Hayoun; Lee, Wu Joo; Lee, Seung-Min

2014-01-01

We aimed to investigate whether in vitro fermentation of soy with L. plantarum could promote its beneficial effects on lipids at the molecular and physiological levels. Rats were fed an AIN76A diet containing 50% sucrose (w/w) (CTRL), a modified AIN76A diet supplemented with 1% (w/w) cholesterol (CHOL), or a CHOL diet where 20% casein was replaced with soy milk (SOY) or fermented soy milk (FSOY). Dietary isoflavone profiles, serum lipids, hepatic and fecal cholesterol, and tissue gene expression were examined. The FSOY diet had more aglycones than did the SOY diet. Both the SOY and FSOY groups had lower hepatic cholesterol and serum triglyceride (TG) than did the CHOL group. Only FSOY reduced hepatic TG and serum free fatty acids and increased serum HDL-CHOL and fecal cholesterol. Compared to CHOL, FSOY lowered levels of the nuclear forms of SREBP-1c and SREBP-2 and expression of their target genes, including FAS, SCD1, LDLR, and HMGCR. On the other hand, FSOY elevated adipose expression levels of genes involved in TG-rich lipoprotein uptake (ApoE, VLDLR, and Lrp1), fatty acid oxidation (PPARα, CPT1α, LCAD, CYP4A1, UCP2, and UCP3), HDL-biogenesis (ABCA1, ApoA1, and LXRα), and adiponectin signaling (AdipoQ, AdipoR1, and AdipoR2), as well as levels of phosphorylated AMPK and ACC. SOY conferred a similar expression profile in both liver and adipose tissues but failed to reach statistical significance in many of the genes tested, unlike FSOY. Our data indicate that fermentation may be a way to enhance the beneficial effects of soy on lipid metabolism, in part via promoting a reduction of SREBP-dependent cholesterol and TG synthesis in the liver, and enhancing adiponectin signaling and PPARα-induced expression of genes involved in TG-rich lipoprotein clearance, fatty acid oxidation, and reverse cholesterol transport in adipose tissues. PMID:24520358

Candidate gene association analysis for milk yield, composition, urea nitrogen and somatic cell scores in Brown Swiss cows.

PubMed

Cecchinato, A; Ribeca, C; Chessa, S; Cipolat-Gotet, C; Maretto, F; Casellas, J; Bittante, G

2014-07-01

The aim of this study was to investigate 96 single-nucleotide polymorphisms (SNPs) from 54 candidate genes, and test the associations of the polymorphic SNPs with milk yield, composition, milk urea nitrogen (MUN) content and somatic cell score (SCS) in individual milk samples from Italian Brown Swiss cows. Milk and blood samples were collected from 1271 cows sampled once from 85 herds. Milk production, quality traits (i.e. protein, casein, fat and lactose percentages), MUN and SCS were measured for each milk sample. Genotyping was performed using a custom Illumina VeraCode GoldenGate approach. A Bayesian linear animal model that considered the effects of herd, days in milk, parity, SNP genotype and additive polygenic effect was used for the association analysis. Our results showed that 14 of the 51 polymorphic SNPs had relevant additive effects on at least one of the aforementioned traits. Polymorphisms in the glucocorticoid receptor DNA-binding factor 1 (GRLF1), prolactin receptor (PRLR) and chemokine ligand 2 (CCL2) were associated with milk yield; an SNP in the stearoyl-CoA desaturase (SCD-1) was related to fat content; SNPs in the caspase recruitment domain 15 protein (CARD15) and lipin 1 (LPIN1) affected the protein and casein contents; SNPs in growth hormone 1 (GH1), lactotransferrin (LTF) and SCD-1 were relevant for casein number; variants in beta casein (CSN2), GH1, GRLF1 and LTF affected lactose content; SNPs in beta-2 adrenergic receptor (ADRB2), serpin peptidase inhibitor (PI) and SCD-1 were associated with MUN; and SNPs in acetyl-CoA carboxylase alpha (ACACA) and signal transducer and activator of transcription 5A (STAT5A) were relevant in explaining the variation of SCS. Although further research is needed to validate these SNPs in other populations and breeds, the association between these markers and milk yield, composition, MUN and SCS could be exploited in gene-assisted selection programs for genetic improvement purposes.

Whey and Casein Proteins and Medium-Chain Saturated Fatty Acids from Milk Do Not Increase Low-Grade Inflammation in Abdominally Obese Adults.

PubMed

Bohl, Mette; Bjørnshave, Ann; Gregersen, Søren; Hermansen, Kjeld

2016-01-01

Low-grade inflammation is involved in the development of diabetes and cardiovascular disease (CVD). Inflammation can be modulated by dietary factors. Dairy products are rich in saturated fatty acids (SFA), which are known to possess pro-inflammatory properties. However, different fatty acid compositions may exert different effects. Other components such as milk proteins may exert anti-inflammatory properties which may compensate for the potential negative effects of SFAs. Generally, the available data suggest a neutral role of dairy product consumption on inflammation. To investigate the effects of, and potential interaction between, a dietary supplementation with whey protein and milk fat, naturally enriched in medium-chain SFA (MC-SFA), on inflammatory markers in abdominal obese adults. The study was a 12-week, randomized, double-blinded, intervention study. Sixty-three adults were equally allocated to one of four groups which received a supplement of either 60 g/day whey or 60 g/day casein plus 63 g/day milk fat either high or low in MC-SFA content. Fifty-two subjects completed the study. Before and after the intervention, changes in plasma interleukin-6 (IL-6), interleukin-1 receptor antagonist (IL-1RA), high-sensitive C-reactive protein (hsCRP), adiponectin, and monocyte chemoattractant protein-1 (MCP-1) were measured. Changes in inflammatory genes in the subcutaneous adipose tissue were also documented. There were no differences in circulating inflammatory markers between protein types or fatty acid compositions in abdominally obese subjects, with the exception of an increase in adiponectin in response to high compared to low MC-SFA consumption in women. We found that combined dairy proteins and MC-SFAs influenced inflammatory gene expression in adipose tissue, while no effect was detected by dairy proteins or MC-SFA per se. Whey protein compared with casein and MC-SFA-enriched milk fat did not alter circulating markers of low-grade inflammation in abdominally obese subjects, except for an increase in circulating adiponectin in response to high MC-SFA in abdominally obese women.

Mammary gland-specific nuclear factor activity is positively regulated by lactogenic hormones and negatively by milk stasis.

PubMed

Schmitt-Ney, M; Happ, B; Hofer, P; Hynes, N E; Groner, B

1992-12-01

The mammary gland-specific nuclear factor (MGF) is a crucial contributor to the regulation of transcription from the beta-casein gene promoter. The beta-casein gene encodes a major milk protein, which is expressed in mammary epithelial cells during lactation and can be induced by lactogenic hormones in the clonal mammary epithelial cell line HC11. We have investigated the specific DNA-binding activity of MGF in mammary epithelial cells in vivo and in vitro. Comparison of MGF in HC11 cells and mammary gland cells from lactating mice revealed molecules with identical DNA-binding properties. Bandshift and UV cross-linking experiments indicated that MGF in HC11 cells has a higher mol wt than MGF found in mice. Little MGF activity was detected in nuclear extracts from HC11 cells cultured in the absence of lactogenic hormones. Lactogenic hormone treatment of HC11 cells led to a strong induction of MGF activity. The induction of MGF activity as well as utilization of the beta-casein promoter were suppressed when epidermal growth factor was present in the tissue culture medium simultaneously with the lactogenic hormones. In lactating animals, MGF activity is regulated by suckling, milk stasis, and systemic hormone signals. The mammary glands from maximally lactating animals, 16 days postpartum, contain drastically reduced MGF activity after removal of the pups for only 8 h. The down-regulation of MGF by pup withdrawal was slower in early lactation, 6 days postpartum. We also investigated the relative contributions of local signals, generated by milk stasis, and systemic hormone signals to the regulation of MGF activity. The access to one row of mammary glands of lactating mothers was denied to the pups for 24 h. High levels of MGF were found in the accessible mammary glands, and intermediate levels of MGF were found in the inaccessible glands of the same mouse. Very low MGF levels were detected when the pups were removed from the dams for 24 h. We conclude that systemic as well as local signals cooperate in the in vitro regulation of MGF activity.

Lupin protein isolate versus casein modifies cholesterol excretion and mRNA expression of intestinal sterol transporters in a pig model

PubMed Central

2014-01-01

Background Lupin proteins exert hypocholesterolemic effects in man and animals, although the underlying mechanism remains uncertain. Herein we investigated whether lupin proteins compared to casein modulate sterol excretion and mRNA expression of intestinal sterol transporters by use of pigs as an animal model with similar lipid metabolism as humans, and cellular cholesterol-uptake by Caco-2 cells. Methods Two groups of pigs were fed cholesterol-containing diets with either 230 g/kg of lupin protein isolate from L. angustifolius or 230 g/kg casein, for 4 weeks. Faeces were collected quantitatively over a 5 d period for analysis of neutral sterols and bile acids by gas chromatographically methods. The mRNA abundances of intestinal lipid transporters were analysed by real-time RT-PCR. Cholesterol-uptake studies were performed with Caco-2 cells that were incubated with lupin conglutin γ, phytate, ezetimibe or albumin in the presence of labelled [4-14C]-cholesterol. Results Pigs fed the lupin protein isolate revealed lower cholesterol concentrations in total plasma, LDL and HDL than pigs fed casein (P < 0.05). Analysis of faeces revealed a higher output of cholesterol in pigs that were fed lupin protein isolate compared to pigs that received casein (+57.1%; P < 0.05). Relative mRNA concentrations of intestinal sterol transporters involved in cholesterol absorption (Niemann-Pick C1-like 1, scavenger receptor class B, type 1) were lower in pigs fed lupin protein isolate than in those who received casein (P < 0.05). In vitro data showed that phytate was capable of reducing the uptake of labelled [4-14C]-cholesterol into the Caco-2 cells to the same extend as ezetimibe when compared to control (−20.5% vs. −21.1%; P < 0.05). Conclusions Data reveal that the cholesterol-lowering effect of lupin protein isolate is attributable to an increased faecal output of cholesterol and a reduced intestinal uptake of cholesterol. The findings indicate phytate as a possible biofunctional ingredient of lupin protein isolate. PMID:24490902

Renoprotective and blood pressure-lowering effect of dietary soy protein via protein kinase C beta II inhibition in a rat model of metabolic syndrome.

PubMed

Palanisamy, Nallasamy; Viswanathan, Periyasamy; Ravichandran, Mambakkam Katchapeswaran; Anuradha, Carani Venkataraman

2010-01-01

We studied whether substitution of soy protein for casein can improve insulin sensitivity, lower blood pressure (BP), and inhibit protein kinase C betaII (PKCbetaII) activation in kidney in an acquired model of metabolic syndrome. Adult male rats were fed 4 different diets: (i) starch (60%) and casein (20%) (CCD), (ii) fructose (60%) and casein (20%) (FCD), (iii) fructose (60%) and soy protein (20%) (FSD), and (iv) starch (60%) and soy protein (20%) (CSD). Renal function parameters, BP, pressor mechanisms, PKCbetaII expression, oxidative stress, and renal histology were evaluated after 60 days. FCD rats displayed insulin resistance and significant changes in body weight, kidney weight, urine volume, plasma and urine electrolytes accompanied by significant changes in renal function parameters compared with CCD rats. Elevated BP, plasma angiotensin-converting enzyme (ACE) activity, renal oxidative stress, and reduced nitrite (NO) and kallikrein activity were observed. Western blot analysis revealed enhanced renal expression of membrane-associated PKCbetaII in the FCD group. Histology showed fatty infiltration and thickening of glomeruli while urinary protein profile revealed a 5-fold increase in albumin. Substitution of soy protein for casein improved insulin sensitivity, lowered BP and PKCbetaII activation and restored renal function. Antioxidant action, inhibitory effect on ACE and PKCbetaII activation, and increased availability of kinins and NO could be contributing mechanisms for the benefits of dietary soy protein.

Glutamine and ornithine alpha-ketoglutarate supplementation on malate dehydrogenases expression in hepatectomized rats.

PubMed

Guimarães Filho, Artur; Cunha, Rodrigo Maranguape Silva da; Vasconcelos, Paulo Roberto Leitão de; Guimarães, Sergio Botelho

2014-06-01

To evaluate the relative gene expression (RGE) of cytosolic (MDH1) and mitochondrial (MDH2) malate dehydrogenases enzymes in partially hepatectomized rats after glutamine (GLN) or ornithine alpha-ketoglutarate (OKG) suplementation. One-hundred and eight male Wistar rats were randomly distributed into six groups (n=18): CCaL, GLNL and OKGL and fed calcium caseinate (CCa), GLN and OKG, 0.5 g/Kg by gavage, 30 minutes before laparotomy. CCaH, GLNH and OKGH groups were likewise fed 30 minutes before 70% partial hepatectomy. Blood and liver samples were collected three, seven and 14 days after laparotomy/hepatectomy for quantification of MDH1/MDH2 enzymes using the real-time polymerase chain reaction (PCR) methodology. Relative enzymes expression was calculated by the 2-(ΔΔC)T method using the threshold cycle (CT) value for normalization. MDH1/MDH2 RGE was not different in hepatectomized rats treated with OKG compared to rats treated with CCa. However, MDH1/MDH2 RGE was greater on days 3 (321:1/26.48:1) and 7 (2.12:1/2.48:1) while MDH2 RGE was greater on day 14 (7.79:1) in hepatectomized rats treated with GLN compared to control animals. Glutamine has beneficial effects in liver regeneration in rats by promoting an up-regulation of the MDH1 and MDH2 relative gene expression.

Prokaryotic expression of bone sialoprotein and identification of casein kinase II phosphorylation sites.

PubMed

Saad, Fawzy A; Salih, Erdjan; Wunderlich, Livius; Flückiger, Rudolf; Glimcher, Melvin J

2005-07-29

Bone sialoprotein is an extracellular noncollagenous acidic protein that plays a role in bone mineralization and remodeling. Its expression is restricted to mineralized tissues and is subjected to variety of posttranslational modifications including phosphorylation and glycosylation. We have expressed the full-length and half domains of bovine bone sialoprotein in a prokaryotic system and identified the phosphorylation sites of casein kinase II. The N-terminal automated solid-phase sequencing defined four phosphorylated peptides: residues 28-38 (LEDS(P)EENGVFK), 51-86 (FYPELKRFAVQSSS(P)DS(P)S(P)EENGNGDS(P)S(P)EEEEEEEETS(P)), 151-165 (EDES(P)DEEEEEEEEEE), and 295-305 (GRGYDS(P)YDGQD). Nine phosphoserines were identified within the four peptides. Seven of them were in the N-terminus (S31, S64, S66, S67, S75, S76, and S86) and two were in the C-terminus (S154 and S300) of the protein.

Choline Transport Activity Regulates Phosphatidylcholine Synthesis through Choline Transporter Hnm1 Stability*

PubMed Central

Fernández-Murray, J. Pedro; Ngo, Michael H.; McMaster, Christopher R.

2013-01-01

Choline is a precursor for the synthesis of phosphatidylcholine through the CDP-choline pathway. Saccharomyces cerevisiae expresses a single high affinity choline transporter at the plasma membrane, encoded by the HNM1 gene. We show that exposing cells to increasing levels of choline results in two different regulatory mechanisms impacting Hnm1 activity. Initial exposure to choline results in a rapid decrease in Hnm1-mediated transport at the level of transporter activity, whereas chronic exposure results in Hnm1 degradation through an endocytic mechanism that depends on the ubiquitin ligase Rsp5 and the casein kinase 1 redundant pair Yck1/Yck2. We present details of how the choline transporter is a major regulator of phosphatidylcholine synthesis. PMID:24187140

Caloric stress alters fat characteristics and Hsp70 expression in milk somatic cells of lactating beef cows

PubMed Central

Eitam, Harel; Brosh, Arieh; Orlov, Alla; Izhaki, Ido

2008-01-01

Selection for higher production rate in cattle inhabiting challenging habitats may be considered disadvantageous because of possible deleterious effects on immunity and reproduction and, consequently, on calf crop percentage. In Israel, free-grazing high productive beef cows experience reduction in nutritional quality of forage during up to 8 months of the year. As milk production by dams dictates calf performance, dam’s nutritional needs and rebreeding rates, the aim of the present study was to test how lactating beef cows deal with combined caloric and protein stress both at the productive and self protective levels. For this purpose, we studied the effect of long-term caloric stress on milk characteristics and gene expression of stress and milk components producing proteins. Lactating dams responded to caloric stress by decreased body weight, milk, and milk protein production. To compensate for total energy loses in milk, they produced milk of higher fat concentration and shifted the proportions of its fatty acids towards long and unsaturated ones. This was reflected by increased mRNA transcription of the fatty acid binding protein. Prolonged low-energy diet promoted cell-specific heat shock protein (Hsp) response; whereas significant increase of Hsp90 but unchanged levels of Hsp70 proteins were observed in white blood cells, the expression of Hsp70 in milk somatic cells was markedly attenuated, in parallel with a marked increase of αs1-casein expression. At the mammary gland level, these results may indicate a decrease in turnover of proteins and a shift to an exclusive expression of milk components producing factors. Similar responses to caloric stress were revealed also in ketotic dairy cows. Ketosis promoted a shift towards long and unsaturated fatty acids and an increased expression of αs1-casein in milk somatic cells. These findings may reflect an evolutionary-preserved mechanism in lactating cows for coping with caloric restriction. Overall, our results provide an index to test suitability of beef cattle breeds to inadequate caloric demands. PMID:18704763

Dystroglycan loss disrupts polarity and beta-casein induction inmammary epithelial cells by perturbing laminin anchoring

DOE Office of Scientific and Technical Information (OSTI.GOV)

Weir, M. Lynn; Oppizzi, Maria Luisa; Henry, Michael D.

2006-02-17

Precise contact between epithelial cells and their underlying basement membrane is critical to the maintenance of tissue architecture and function. To understand the role that the laminin receptor dystroglycan (DG) plays in these processes, we assayed cell responses to laminin-111 following conditional ablation of DG expression in cultured mammary epithelial cells (MECs). Strikingly, DG loss disrupted laminin-111-induced polarity and {beta}-casein production, and abolished laminin assembly at the step of laminin binding to the cell surface. DG re-expression restored these deficiencies. Investigations of mechanism revealed that DG cytoplasmic sequences were not necessary for laminin assembly and signaling, and only when themore » entire mucin domain of extracellular DG was deleted did laminin assembly not occur. These results demonstrate that DG is essential as a laminin-111 co-receptor in MECs that functions by mediating laminin anchoring to the cell surface, a process that allows laminin polymerization, tissue polarity, and {beta}-casein induction. The observed loss of laminin-111 assembly and signaling in DG-/-MECs provides insights into the signaling changes occurring in breast carcinomas and other cancers, where DG's laminin-binding function is frequently defective.« less

Acquisition and amplification of a testis-expressed autosomal gene, SSL, by the Drosophila Y chromosome

PubMed Central

Kalmykova, Alla I.; Shevelyov, Yury Y.; Dobritsa, Anna A.; Gvozdev, Vladimir A.

1997-01-01

The acquisition of autosomal fertility genes has been proposed to be an important process in human Y chromosome evolution. For example, the Y-linked fertility factor DAZ (Deleted in Azoospermia) appears to have arisen after the transposition and tandem amplification of the autosomal DAZH gene. The Drosophila melanogaster Y chromosome contains tandemly repeated Su(Ste) units that are thought to affect male fertility as suppressors of the homologous X-linked Stellate repeats. Here we report the detection of a testis-expressed autosomal gene, SSL [Su(Ste)-like], that appears to be an ancestor of the Y-linked Su(Ste) units. SSL encodes a casein kinase 2 (CK2) β-subunit-like protein. Its putative ORF shares extensive (45%) homology with the genuine β-subunit of CK2 and retains the conserved C-terminal and Glu/Asp-rich domains that are essential for CK2 holoenzyme regulation. SSL maps within region 60D1–2 of D. melanogaster and D. simulans polytene chromosomes. We present evidence that SSL was derived from the genuine βCK2 gene by reverse transcription. This event resulted in the loss of the first three introns in the coding region of the SSL ancestor gene. Evolutionary analysis indicates that SSL has evolved under selective pressure at the translational level. Its sequence, especially in the 3′ region, is much closer to the Y-linked Su(Ste) tandem repeats than to the βCK2 gene. These results suggest that the acquisition of testis-specific autosomal genes may be important for the evolution of Drosophila as well as human Y chromosomes. PMID:9177211

Uterine responses to feeding soy protein isolate and treatment with 17β-estradiol differ in ovariectomized female rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ronis, Martin J., E-mail: mronis@lsuhsc.edu; Gomez-Acevedo, Horacio; Blackburn, Michael L.

There are concerns regarding reproductive toxicity from consumption of soy foods, including an increased risk of endometriosis and endometrial cancer, as a result of phytoestrogen consumption. In this study, female rats were fed AIN-93G diets made with casein (CAS) or soy protein isolate (SPI) from postnatal day (PND) 30, ovariectomized on PND 50 and infused with 5 μg/kg/d 17β-estradiol (E2) or vehicle. E2 increased uterine wet weight (P < 0.05). RNAseq analysis revealed that E2 significantly altered expression of 1991 uterine genes (P < 0.05). SPI feeding had no effect on uterine weight and altered expression of far fewer genesmore » than E2 at 152 genes (P < 0.05). Overlap between E2 and SPI genes was limited to 67 genes. Functional annotation analysis indicated significant differences in uterine biological processes affected by E2 and SPI and little evidence for recruitment of estrogen receptor (ER)α to the promoters of ER-responsive genes after SPI feeding. The major E2 up-regulated uterine pathways were carcinogenesis and extracellular matrix organization, whereas SPI feeding up-regulated uterine peroxisome proliferator activated receptor (PPAR) signaling and fatty acid metabolism. The combination of E2 and SPI resulted in significant regulation of 504 fewer genes relative to E2 alone. The ability of E2 to induce uterine proliferation in response to the carcinogen dimethybenz(a)anthracene (DMBA) as measured by expression of PCNA and Ki67 mRNA was suppressed by feeding SPI (P < 0.05). These data suggest that SPI is a selective estrogen receptor modulator (SERM) interacting with a small sub-set of E2-regulated genes and is anti-estrogenic in the presence of endogenous estrogens. - Highlights: • Concerns exist regarding risk of uterine cancer from consumption of soy products. • These concerns are related to potential estrogenicity. • Estradiol and soy protein isolate effects on uterine gene expression were compared. • Soy acts as a selective estrogen receptor modulator not a weak estrogen. • Soy feeding blocked uterine proliferation after treatment with estradiol and DMBA.« less

Mammary cell-activating factor regulates the hormone-independent transcription of the early lactation protein (ELP) gene in a marsupial.

PubMed

Pharo, Elizabeth A; Renfree, Marilyn B; Cane, Kylie N

2016-11-15

The regulation of the tammar wallaby (Macropus eugenii) early lactation protein (ELP) gene is complex. ELP is responsive to the lactogenic hormones; insulin (I), hydrocortisone (HC) and prolactin (PRL) in mammary gland explants but could not be induced with lactogenic hormones in tammar primary mammary gland cells, nor in KIM-2 conditionally immortalised murine mammary epithelial cells. Similarly, ELP promoter constructs transiently-transfected into human embryonic kidney (HEK293T) cells constitutively expressing the prolactin receptor (PRLR) and Signal Transducer and Activator of Transcription (STAT)5A were unresponsive to prolactin, unlike the rat and mouse β-casein (CSN2) promoter constructs. Identification of the minimal promoter required for the hormone-independent transcription of tammar ELP in HEK293Ts and comparative analysis of the proximal promoters of marsupial ELP and the orthologous eutherian colostrum trypsin inhibitor (CTI) gene suggests that mammary cell-activating factor (MAF), an E26 transformation-specific (ETS) factor, may bind to an AGGAAG motif and activate tammar ELP. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Dietary Mung Bean Protein Reduces Hepatic Steatosis, Fibrosis, and Inflammation in Male Mice with Diet-Induced, Nonalcoholic Fatty Liver Disease.

PubMed

Watanabe, Hitoshi; Inaba, Yuka; Kimura, Kumi; Asahara, Shun-Ichiro; Kido, Yoshiaki; Matsumoto, Michihiro; Motoyama, Takayasu; Tachibana, Nobuhiko; Kaneko, Shuichi; Kohno, Mitsutaka; Inoue, Hiroshi

2017-01-01

As the prevalence of nonalcoholic fatty liver disease (NAFLD), including steatosis and nonalcoholic steatohepatitis, is increasing, novel dietary approaches are required for the prevention and treatment of NAFLD. We evaluated the potential of mung bean protein isolate (MuPI) to prevent NAFLD progression. In Expts. 1 and 2, the hepatic triglyceride (TG) concentration was compared between 8-wk-old male mice fed a high-fat diet (61% of energy from fat) containing casein, MuPI, and soy protein isolate and an MuPI-constituent amino acid mixture as a source of amino acids (18% of energy) for 4 wk. In Expt. 3, hepatic fatty acid synthase (Fasn) expression was evaluated in 8-wk-old male Fasn-promoter-reporter mice fed a casein- or MuPI-containing high-fat diet for 20 wk. In Expt. 4, hepatic fibrosis was examined in 8-wk-old male mice fed an atherogenic diet (61% of energy from fat, containing 1.3 g cholesterol/100 g diet) containing casein or MuPI (18% of energy) as a protein source for 20 wk. In the high fat-diet mice, the hepatic TG concentration in the MuPI group decreased by 66% and 47% in Expt. 1 compared with the casein group (P < 0.001) and the soy protein isolate group (P = 0.001), respectively, and decreased by 56% in Expt. 2 compared with the casein group (P = 0.011). However, there was no difference between the MuPI-constituent amino acid mixture and casein groups in Expt. 2. In Expt. 3, Fasn-promoter-reporter activity and hepatic TG concentration were lower in the MuPI group than in those fed casein (P < 0.05). In Expt. 4, in mice fed an atherogenic diet, hepatic fibrosis was not induced in the MuPI group, whereas it developed overtly in the casein group. MuPI potently reduced hepatic lipid accumulation in mice and may be a potential foodstuff to prevent NAFLD onset and progression. © 2017 American Society for Nutrition.

Protein composition of different sized casein micelles in milk after the binding of lactoferrin or lysozyme.

PubMed

Anema, Skelte G; de Kruif, C G Kees

2013-07-24

Casein micelles with bound lactoferrin or lysozyme were fractionated into sizes ranging in radius from ∼50 to 100 nm. The κ-casein content decreased markedly and the αS-casein/β-casein content increased slightly as micelle size increased. For lactoferrin, higher levels were bound to smaller micelles. The lactoferrin/κ-casein ratio was constant for all micelle sizes, whereas the lactoferrin/αS-casein and lactoferrin/β-casein ratio decreased with increasing micelle size. This indicates that the lactoferrin was binding to the surface of the casein micelles. For lysozyme, higher levels bound to larger casein micelles. The lysozyme/αS-casein and lysozyme/β-casein ratios were nearly constant, whereas the lysozyme/κ-casein ratio increased with increasing micelle size, indicating that lysozyme bound to αS-casein and β-casein in the micelle core. Lactoferrin is a large protein that cannot enter the casein protein mesh; therefore, it binds to the micelle surface. The smaller lysozyme can enter the protein mesh and therefore binds to the more charged αS-casein and β-casein.

Simultaneous Presence of PrtH and PrtH2 Proteinases in Lactobacillus helveticus Strains Improves Breakdown of the Pure αs1-Casein▿ †

PubMed Central

Sadat-Mekmene, L.; Jardin, J.; Corre, C.; Mollé, D.; Richoux, R.; Delage, M.-M.; Lortal, S.; Gagnaire, V.

2011-01-01

Lactobacillus helveticus can possess one or two cell envelope proteinases (CEPs), called PrtH2 and PrtH. The aim of this work was to explore the diversity of 15 strains of L. helveticus, isolated from various origins, in terms of their proteolytic activities and specificities on pure caseins or on milk casein micelles. CEP activity differed 14-fold when the strains were assayed on a synthetic substrate, but no significant differences were detected between strains possessing one or two CEPs. No correlation was observed between the proteolytic activities of the strains and their rates of acidification in milk. The kinetics of hydrolysis of purified αs1- and β-casein by L. helveticus whole cells was monitored using Tris-Tricine sodium dodecyl sulfate (SDS) electrophoresis, and for four strains, the peptides released were identified using mass spectrometry. While rapid hydrolysis of pure β-casein was observed for all strains, the hydrolysis kinetics of αs1-casein was the only criterion capable of distinguishing between the strains based on the number of CEPs. Fifty-four to 74 peptides were identified for each strain. When only PrtH2 was present, 22 to 30% of the peptides originated from αs1-casein. The percentage increased to 41 to 49% for strains in which both CEPs were expressed. The peptide size ranged from 6 to 33 amino acids, revealing a broad range of cleavage specificities, involving all classes of amino acids (Leu, Val, Ala, Ile, Glu, Gln, Lys, Arg, Met, and Pro). Regions resistant to proteolysis were identified in both caseins. When strains were grown in milk, a drastic reduction in the number of peptides was observed, reflecting changes in accessibility and/or peptide assimilation during growth. PMID:21037305

Casein polymorphism heterogeneity influences casein micelle size in milk of individual cows.

PubMed

Day, L; Williams, R P W; Otter, D; Augustin, M A

2015-06-01

Milk samples from individual cows producing small (148-155 nm) or large (177-222 nm) casein micelles were selected to investigate the relationship between the individual casein proteins, specifically κ- and β-casein phenotypes, and casein micelle size. Only κ-casein AA and β-casein A1A1, A1A2 and A2A2 phenotypes were found in the large casein micelle group. Among the small micelle group, both κ-casein and β-casein phenotypes were more diverse. κ-Casein AB was the dominant phenotype, and 3 combinations (AA, AB, and BB) were present in the small casein micelle group. A considerable mix of β-casein phenotypes was found, including B and I variants, which were only found in the small casein micelle group. The relative amount of κ-casein to total casein was significantly higher in the small micelle group, and the nonglycosylated and glycosylated κ-casein contents were higher in the milks with small casein micelles (primarily with κ-casein AB and BB variants) compared with the large micelle group. The ratio of glycosylated to nonglycosylated κ-casein was higher in the milks with small casein micelles compared with the milks with large casein micelles. This suggests that although the amount of κ-casein (both glycosylated and nonglycosylated) is associated with micelle size, an increased proportion of glycosylated κ-casein could be a more important and favorable factor for small micelle size. This suggests that the increased spatial requirement due to addition of the glycosyl group with increasing extent of glycosylation of κ-casein is one mechanism that controls casein micelle assembly and growth. In addition, increased electrostatic repulsion due to the sialyl residues on the glycosyl group could be a contributory factor. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Cu/Zn superoxide dismutase and the proton ATPase Pma1p of Saccharomyces cerevisiae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baron, J. Allen; Chen, Janice S.; Culotta, Valeria C., E-mail: vculott1@jhu.edu

2015-07-03

In eukaryotes, the Cu/Zn containing superoxide dismutase (SOD1) plays a critical role in oxidative stress protection as well as in signaling. We recently demonstrated a function for Saccharomyces cerevisiae Sod1p in signaling through CK1γ casein kinases and identified the essential proton ATPase Pma1p as one likely target. The connection between Sod1p and Pma1p was explored further by testing the impact of sod1Δ mutations on cells expressing mutant alleles of Pma1p that alter activity and/or post-translational regulation of this ATPase. We report here that sod1Δ mutations are lethal when combined with the T912D allele of Pma1p in the C-terminal regulatory domain.more » This “synthetic lethality” was reversed by intragenic suppressor mutations in Pma1p, including an A906G substitution that lies within the C-terminal regulatory domain and hyper-activates Pma1p. Surprisingly the effect of sod1Δ mutations on Pma1-T912D is not mediated through the Sod1p signaling pathway involving the CK1γ casein kinases. Rather, Sod1p sustains life of cells expressing Pma1-T912D through oxidative stress protection. The synthetic lethality of sod1Δ Pma1-T912D cells is suppressed by growing cells under low oxygen conditions or by treatments with manganese-based antioxidants. We now propose a model in which Sod1p maximizes Pma1p activity in two ways: one involving signaling through CK1γ casein kinases and an independent role for Sod1p in oxidative stress protection. - Highlights: • In yeast, the anti-oxidant enzyme SOD1 promotes activity of the proton ATPase Pma1p. • Cells expressing a T912D variant of Pma1p are not viable without SOD1. • SOD1 is needed to protect Pma1-T912D expressing cells from severe oxidative damage. • SOD1 activates Pma1p through casein kinase signaling and oxidative stress protection.« less

Identification and Partial Characterization of Extracellular Aspartic Protease Genes from Metschnikowia pulcherrima IWBT Y1123 and Candida apicola IWBT Y1384

PubMed Central

Reid, Vernita J.; Theron, Louwrens W.; du Toit, Maret

2012-01-01

The extracellular acid proteases of non-Saccharomyces wine yeasts may fulfill a number of roles in winemaking, which include increasing the available nitrogen sources for the growth of fermentative microbes, affecting the aroma profile of the wine, and potentially reducing protein haze formation. These proteases, however, remain poorly characterized, especially at genetic level. In this study, two extracellular aspartic protease-encoding genes were identified and sequenced, from two yeast species of enological origin: one gene from Metschnikowia pulcherrima IWBT Y1123, named MpAPr1, and the other gene from Candida apicola IWBT Y1384, named CaAPr1. In silico analysis of these two genes revealed a number of features peculiar to aspartic protease genes, and both the MpAPr1 and CaAPr1 putative proteins showed homology to proteases of yeast genera. Heterologous expression of MpAPr1 in Saccharomyces cerevisiae YHUM272 confirmed that it encodes an aspartic protease. MpAPr1 production, which was shown to be constitutive, and secretion were confirmed in the presence of bovine serum albumin (BSA), casein, and grape juice proteins. The MpAPr1 gene was found to be present in 12 other M. pulcherrima strains; however, plate assays revealed that the intensity of protease activity was strain dependent and unrelated to the gene sequence. PMID:22820332

Effect of temperature and pH on the solubility of caseins: environmental influences on the dissociation of α(S)- and β-casein.

PubMed

Post, A E; Arnold, B; Weiss, J; Hinrichs, J

2012-04-01

Selective precipitation is a common method for the isolation of β-casein, using the different calcium sensitivities of the individual caseins and the selective solubility of β-casein at a low temperature. In previous studies, it has been indicated that the β-casein yield depends on the physicochemical characteristics of the casein raw material used for fractionation. The objective of this study was to evaluate and compare the solubility of α(S)- and β-casein in solutions of micellar casein, sodium caseinate, and calcium caseinate as a function of pH and temperature. Additionally, the solubility of isolated α(S)- and β-casein fractions in demineralized water, ultrafiltration permeate, and a calcium-depleted milk salt solution was investigated depending on the pH and temperature. Furthermore, micellar casein, sodium caseinate, and calcium caseinate were subjected to a calcium chloride-precipitation process to determine the solubility of α(S)- and β-casein in calcium chloride precipitate, which is produced during selective precipitation, as a function of temperature and pH. Generally, the temperature had only a marginal influence on the α(S)-casein solubility compared with the β-casein solubility, whereas the solubility was shown to be strongly influenced by the pH. Our results suggest that the yield of β-casein obtained during isolation by means of selective precipitation may be a result of the solubility characteristics of α(S)- and β-casein in calcium chloride precipitate. Manufacturers may consider a simple solubility experiment before the β-casein isolation process by means of selective precipitation to predict β-casein yield. Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

The PA influenza virus polymerase subunit is a phosphorylated protein.

PubMed

Sanz-Ezquerro, J J; Fernández Santarén, J; Sierra, T; Aragón, T; Ortega, J; Ortín, J; Smith, G L; Nieto, A

1998-03-01

The induction of proteolysis by expression of the influenza virus PA polymerase subunit is the only biochemical activity ascribed to this protein. In the course of studying viral protein synthesis by two-dimensional gel electrophoresis, we observed the existence of several PA isoforms with different isoelectric points. These isoforms were also present when the PA gene was singly expressed in three different expression systems, indicating that a cellular activity is responsible for its post-translational modification. In vivo labelling with [32P]orthophosphate, followed by two-dimensional gel electrophoresis, clearly demonstrated the incorporation of phosphate into the PA molecule. Phosphoserine and phosphothreonine epitopes were present in PA, while phosphotyrosine residues were absent, as tested by immunoblotting with specific antibodies. These facts, as well as the presence of multiple consensus sites for casein kinase II (CKII) phosphorylation, prompted us to test the involvement of this kinase in PA covalent modification. PA protein purified by immunoprecipitation could be specifically labelled by the catalytic alpha subunit of human CKII, which was expressed and purified from bacteria. Collectively, these data demonstrate that the PA subunit of the influenza virus RNA polymerase is a phosphoprotein.

Binding sites of resveratrol, genistein, and curcumin with milk α- and β-caseins.

PubMed

Bourassa, P; Bariyanga, J; Tajmir-Riahi, H A

2013-02-07

The binding sites of antioxidant polyphenols resveratrol, genistein, and curcumin are located with milk α- and β-caseins in aqueous solution. FTIR, CD, and fluorescence spectroscopic methods and molecular modeling were used to analyze polyphenol binding sites, the binding constant, and the effects of complexation on casein stability and conformation. Structural analysis showed that polyphenols bind casein via hydrophilic and hydrophobic interactions with the number of bound polyphenol molecules (n) 1.20 for resveratrol, 1.42 for genistein, and 1.43 for curcumin with α-casein and 1.14 for resveratrol, 1.27 for genistein, and 1.27 for curcumin with β-casein. The overall binding constants of the complexes formed are K(res-α-casein) = 1.9 (±0.6) × 10(4) M(-1), K(gen-α-casein) = 1.8 (±0.4) × 10(4) M(-1), and K(cur-α-casein) = 2.8 (±0.8) × 10(4) M(-1) with α-casein and K(res-β-casein) = 2.3 (±0.3) × 10(4) M(-1), K(gen-β-casein) = 3.0 (±0.5) × 10(4) M(-1), and K(cur-β-casein) = 3.1 (±0.5) × 10(4) M(-1) for β-casein. Molecular modeling showed the participation of several amino acids in polyphenol-protein complexes, which were stabilized by the hydrogen bonding network with the free binding energy of -11.56 (resveratrol-α-casein), -12.35 (resveratrol-β-casein), -9.68 (genistein-α-casein), -9.97 (genistein-β-casein), -8.89 (curcumin-α-casein), and -10.70 kcal/mol (curcumin-β-casein). The binding sites of polyphenols are different with α- and β-caseins. Polyphenol binding altered casein conformation with reduction of α-helix, indicating a partial protein destabilization. Caseins might act as carriers to transport polyphenol in vitro.

Comparison of the orogenic displacement of sodium caseinate with the caseins from the air-water interface by nonionic surfactants.

PubMed

Woodward, N C; Gunning, A P; Mackie, A R; Wilde, P J; Morris, V J

2009-06-16

Displacement of sodium caseinate from the air-water interface by nonionic surfactants Tween 20 and Tween 60 was observed by atomic force microscopy (AFM). The interfacial structure was sampled by Langmuir-Blodgett deposition onto freshly cleaved mica substrates. Protein displacement occurred through an orogenic mechanism: it involved the nucleation and growth of surfactant domains within the protein network, followed by failure of the protein network. The surface pressure at which failure of the protein network occurred was essentially independent of the type of surfactant. The major component of sodium caseinate is beta-casein, and previous studies at the air-water interface have shown that beta-casein networks are weak, failing at surface pressures below that observed for sodium caseinate. The other components of sodium caseinate are alpha(s)- and kappa-caseins. Studies of the displacement of alpha(s)-caseins from air-water interfaces show that these proteins also form weak networks that fail at surface pressures below that observed for sodium caseinate. However, kappa-casein was found to form strong networks that resisted displacement and failed at surface pressures comparable to those observed for sodium caseinate. The AFM images of the displacement suggest that, despite kappa-casein being a minor component, it dominates the failure of sodium caseinate networks: alpha(s)-casein and beta-casein are preferentially desorbed at lower surface pressures, allowing the residual kappa-casein to control the breakdown of the sodium caseinate network at higher surface pressures.

Malaria Parasite-Infected Erythrocytes Secrete PfCK1, the Plasmodium Homologue of the Pleiotropic Protein Kinase Casein Kinase 1

PubMed Central

Dorin-Semblat, Dominique; Demarta-Gatsi, Claudia; Hamelin, Romain; Armand, Florence; Carvalho, Teresa Gil; Moniatte, Marc; Doerig, Christian

2015-01-01

Casein kinase 1 (CK1) is a pleiotropic protein kinase implicated in several fundamental processes of eukaryotic cell biology. Plasmodium falciparum encodes a single CK1 isoform, PfCK1, that is expressed at all stages of the parasite’s life cycle. We have previously shown that the pfck1 gene cannot be disrupted, but that the locus can be modified if no loss-of-function is incurred, suggesting an important role for this kinase in intra-erythrocytic asexual proliferation. Here, we report on the use of parasite lines expressing GFP- or His-tagged PfCK1 from the endogenous locus to investigate (i) the dynamics of PfCK1 localisation during the asexual cycle in red blood cells, and (ii) potential interactors of PfCK1, so as to gain insight into the involvement of the enzyme in specific cellular processes. Immunofluorescence analysis reveals a dynamic localisation of PfCK1, with evidence for a pool of the enzyme being directed to the membrane of the host erythrocyte in the early stages of infection, followed by a predominantly intra-parasite localisation in trophozoites and schizonts and association with micronemes in merozoites. Furthermore, we present strong evidence that a pool of enzymatically active PfCK1 is secreted into the culture supernatant, demonstrating that PfCK1 is an ectokinase. Our interactome experiments and ensuing kinase assays using recombinant PfCK1 to phosphorylate putative interactors in vitro suggest an involvement of PfCK1 in many cellular processes such as mRNA splicing, protein trafficking, ribosomal, and host cell invasion. PMID:26629826

Effect of maternal protein restriction during pregnancy and postweaning high-fat feeding on diet-induced thermogenesis in adult mouse offspring.

PubMed

Sellayah, Dyan; Dib, Lea; Anthony, Frederick W; Watkins, Adam J; Fleming, Tom P; Hanson, Mark A; Cagampang, Felino R

2014-10-01

Prenatal undernutrition followed by postweaning feeding of a high-fat diet results in obesity in the adult offspring. In this study, we investigated whether diet-induced thermogenesis is altered as a result of such nutritional mismatch. Female MF-1 mice were fed a normal protein (NP, 18% casein) or a protein-restricted (PR, 9% casein) diet throughout pregnancy and lactation. After weaning, male offspring of both groups were fed either a high-fat diet (HF; 45% kcal fat) or standard chow (C, 7% kcal fat) to generate the NP/C, NP/HF, PR/C and PR/HF adult offspring groups (n = 7-11 per group). PR/C and NP/C offspring have similar body weights at 30 weeks of age. Postweaning HF feeding resulted in significantly heavier NP/HF offspring (P < 0.01), but not in PR/HF offspring, compared with their chow-fed counterparts. However, the PR/HF offspring exhibited greater adiposity (P < 0.01) v the NP/HF group. The NP/HF offspring had increased energy expenditure and increased mRNA expression of uncoupling protein-1 and β-3 adrenergic receptor in the interscapular brown adipose tissue (iBAT) compared with the NP/C mice (both at P < 0.01). No such differences in energy expenditure and iBAT gene expression were observed between the PR/HF and PR/C offspring. These data suggest that a mismatch between maternal diet during pregnancy and lactation, and the postweaning diet of the offspring, can attenuate diet-induced thermogenesis in the iBAT, resulting in the development of obesity in adulthood.

FAM83H and casein kinase I regulate the organization of the keratin cytoskeleton and formation of desmosomes

PubMed Central

Kuga, Takahisa; Sasaki, Mitsuho; Mikami, Toshinari; Miake, Yasuo; Adachi, Jun; Shimizu, Maiko; Saito, Youhei; Koura, Minako; Takeda, Yasunori; Matsuda, Junichiro; Tomonaga, Takeshi; Nakayama, Yuji

2016-01-01

FAM83H is essential for the formation of dental enamel because a mutation in the FAM83H gene causes amelogenesis imperfecta (AI). We previously reported that the overexpression of FAM83H often occurs and disorganizes the keratin cytoskeleton in colorectal cancer cells. We herein show that FAM83H regulates the organization of the keratin cytoskeleton and maintains the formation of desmosomes in ameloblastoma cells. FAM83H is expressed and localized on keratin filaments in human ameloblastoma cell lines and in mouse ameloblasts and epidermal germinative cells in vivo. FAM83H shows preferential localization to keratin filaments around the nucleus that often extend to cell-cell junctions. Alterations in the function of FAM83H by its overexpression, knockdown, or an AI-causing truncated mutant prevent the proper organization of the keratin cytoskeleton in ameloblastoma cells. Furthermore, the AI-causing mutant prevents desmosomal proteins from being localized to cell-cell junctions. The effects of the AI-causing mutant depend on its binding to and possible inhibition of casein kinase I (CK-1). The suppression of CK-1 by its inhibitor, D4476, disorganizes the keratin cytoskeleton. Our results suggest that AI caused by the FAM83H mutation is mediated by the disorganization of the keratin cytoskeleton and subsequent disruption of desmosomes in ameloblasts. PMID:27222304

FAM83H and casein kinase I regulate the organization of the keratin cytoskeleton and formation of desmosomes.

PubMed

Kuga, Takahisa; Sasaki, Mitsuho; Mikami, Toshinari; Miake, Yasuo; Adachi, Jun; Shimizu, Maiko; Saito, Youhei; Koura, Minako; Takeda, Yasunori; Matsuda, Junichiro; Tomonaga, Takeshi; Nakayama, Yuji

2016-05-25

FAM83H is essential for the formation of dental enamel because a mutation in the FAM83H gene causes amelogenesis imperfecta (AI). We previously reported that the overexpression of FAM83H often occurs and disorganizes the keratin cytoskeleton in colorectal cancer cells. We herein show that FAM83H regulates the organization of the keratin cytoskeleton and maintains the formation of desmosomes in ameloblastoma cells. FAM83H is expressed and localized on keratin filaments in human ameloblastoma cell lines and in mouse ameloblasts and epidermal germinative cells in vivo. FAM83H shows preferential localization to keratin filaments around the nucleus that often extend to cell-cell junctions. Alterations in the function of FAM83H by its overexpression, knockdown, or an AI-causing truncated mutant prevent the proper organization of the keratin cytoskeleton in ameloblastoma cells. Furthermore, the AI-causing mutant prevents desmosomal proteins from being localized to cell-cell junctions. The effects of the AI-causing mutant depend on its binding to and possible inhibition of casein kinase I (CK-1). The suppression of CK-1 by its inhibitor, D4476, disorganizes the keratin cytoskeleton. Our results suggest that AI caused by the FAM83H mutation is mediated by the disorganization of the keratin cytoskeleton and subsequent disruption of desmosomes in ameloblasts.

Anti-inflammatory effects of a casein hydrolysate and its peptide-enriched fractions on TNFα-challenged Caco-2 cells and LPS-challenged porcine colonic explants

PubMed Central

Mukhopadhya, Anindya; Noronha, Nessa; Bahar, Bojlul; Ryan, Marion T; Murray, Brian A; Kelly, Phil M; O'Loughlin, Ian B; O'Doherty, John V; Sweeney, Torres

2014-01-01

Bioactive milk peptides are reported to illicit a range of physiological benefits and have been proposed as potential functional food ingredients. The objective of this study was to characterize the anti-inflammatory properties of sodium caseinate (NaCAS), its enzyme hydrolysate (EH) and peptide-enriched fractions (5 kDa retentate [R], 1 kDaR and 1 kDa permeate [P]), both in vitro using a Caco-2 cell line, and also ex vivo using a porcine colonic tissue explant system. Caco-2 cells were stimulated with tumour necrosis factor alpha (TNFα) and co-treated with casein hydrolysates for 24 h. Following this, interleukin (IL)-8 concentrations in the supernatant were measured using enzyme-linked immunosorbent assay. Porcine colonic tissue was stimulated with lipopolysaccharide and co-treated with casein hydrolysates for 3 h. The expression of a panel of inflammatory cytokines was measured using qPCR. While dexamethasone reduced the IL-8 concentration by 41.6%, the 1 kDaR and 1 kDaP fractions reduced IL-8 by 68.7% and 66.1%, respectively, relative to TNFα-stimulated Caco-2 cells (P < 0.05). In the ex vivo system, only the 1 kDaR fraction elicited a decrease inIL1-α,IL1-β,IL-8,TGF-β andIL-10 expression (P < 0.05). This study provides evidence that the bioactive peptides present in the 1 kDaR fraction of the NaCAS hydrolysate possess anti-inflammatory properties in vitro and ex vivo. Further in vivo analysis of the anti-inflammatory properties of the 1 kDaR is proposed. PMID:25493190

Sensitizing capacity and allergenicity of enzymatically cross-linked sodium caseinate in comparison to sodium caseinate in a mouse model for cow's milk allergy.

PubMed

van Esch, Betty C A M; Gros-van Hest, Marjan; Westerbeek, Hans; Garssen, Johan

2013-03-27

A transglutaminase cross-linked caseinate was designed for use in dairy products to increase the viscosity of food matrices. The difference in structure of cross-linked caseinate might have implications for the risk of developing cow's milk allergy. The sensitizing capacity and the allergenicity (the potency to induce an allergic effector response) of cross-linked sodium caseinate was investigated using a mouse model for cow's milk allergy. Mice were orally sensitized with cross-linked caseinate or caseinate using cholera toxin as adjuvant. Anaphylactic shock reactions, change in body temperature, acute allergic skin response, caseinate-, cross-linked caseinate-IgE and mMCP-1 concentrations were determined after challenge with cross-linked caseinate or caseinate. Sensitization with cross-linked caseinate did not result in anaphylactic shock symptoms, drop in body temperature or release of serum mMCP-1. A tendency toward decreased casein-specific IgE levels was observed. The allergenicity did not differ between both products. These results indicate that in already caseinate-sensitized mice, cross-linked caseinate did not provoke more pronounced allergenic reactions compared to sodium caseinate. On top of that, reduced sensitization to cross-linked caseinate was observed. Cross-linked caseinate might therefore be an interesting new dietary concept for humans at risk for food allergy although more mechanistic studies and clinical trials are needed for validation. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Calcium-containing phosphopeptides pave the secretory pathway for efficient protein traffic and secretion in fungi.

PubMed

Martín, Juan F

2014-09-10

Casein phosphopeptides (CPPs) containing chelated calcium drastically increase the secretion of extracellular homologous and heterologous proteins in filamentous fungi. Casein phosphopeptides released by digestion of alpha - and beta-casein are rich in phosphoserine residues (SerP). They stimulate enzyme secretion in the gastrointestinal tract and enhance the immune response in mammals, and are used as food supplements. It is well known that casein phosphopeptides transport Ca2+ across the membranes and play an important role in Ca2+ homeostasis in the cells. Addition of CPPs drastically increases the production of heterologous proteins in Aspergillus as host for industrial enzyme production. Recent proteomics studies showed that CPPs alter drastically the vesicle-mediated secretory pathway in filamentous fungi, apparently because they change the calcium concentration in organelles that act as calcium reservoirs. In the organelles calcium homeostasis a major role is played by the pmr1 gene, that encodes a Ca2+/Mn2+ transport ATPase, localized in the Golgi complex; this transporter controls the balance between intra-Golgi and cytoplasmic Ca2+ concentrations. A Golgi-located casein kinase (CkiA) governs the ER to Golgi directionality of the movement of secretory proteins by interacting with the COPII coat of secretory vesicles when they reach the Golgi. Mutants defective in the casein-2 kinase CkiA show abnormal targeting of some secretory proteins, including cytoplasmic membrane amino acid transporters that in ckiA mutants are miss-targeted to vacuolar membranes. Interestingly, addition of CPPs increases a glyceraldehyde-3-phpshate dehydrogenase protein that is known to associate with microtubules and act as a vesicle/membrane fusogenic agent. In summary, CPPs alter the protein secretory pathway in fungi adapting it to a deregulated protein traffic through the organelles and vesicles what results in a drastic increase in secretion of heterologous and also of some homologous proteins.

Investigating rennet coagulation properties of recombined highly concentrated micellar casein concentrate and cream for use in cheese making.

PubMed

Lu, Y; McMahon, D J; Vollmer, A H

2017-02-01

Highly concentrated micellar casein concentrate (HC-MCC) contains ∼18% casein with ∼70% of whey proteins removed by microfiltration and diafiltration of skim milk, followed by vacuum evaporation for further concentration. When blended with cream, HC-MCC forms recombined concentrated milk (RCM), which could be used as a starting material in cheese making. Our objective was to investigate the rennet coagulation properties of RCM while varying parameters such as casein level, pH, rennet level, and coagulation temperature. The HC-MCC was mixed with cream using low shear at 50°C for 10 min, followed by cooling to 31, 28, or 25°C and adding rennet, and rheological properties were determined. Rennet coagulation time [RCT, the time at which storage modulus (G') = loss modulus (G″)] decreased from 8.7 to 7.4 min as casein level increased from 3.2 to 5.7%, without a significant additional difference in RCT at casein levels >5.7%. The initial G″ (G″ 0 ) increased about 10-fold when casein levels were increased from 3.2 to 10.9%, whereas no change in initial G' (G' 0 ) was observed. When G' was measured relative to RCT (i.e., 1, 1.5, or 2 times RCT after RCT was reached, and expressed as G' 1 , G' 1.5 , and G' 2 ), log relationship was found between relative G' and casein level (R 2 > 0.94). Lowering coagulation temperature from 31 to 25°C increased G″ 0 by 6 fold and extended RCT from 7.4 to 9.5 min. After coagulation, relative G' was initially higher at the lower temperature with G' 1 of 3.6 Pa at 25°C and 2.0 Pa at 31°C, but delayed in further development with G' 2 of 0.8 kPa at 25°C and 1.1 kPa at 31°C. Lowering pH of RCM from 6.6 to 6.2 resulted in reduced RCT from 11.9 to 6.5 min with increased relative G' after coagulation. When less rennet was used, RCT increased in a linear inverse relationship without changes in relative G' or G″. The microstructure of RCM coagulum (∼11% casein), observed using transmission electron microscopy, confirmed that RCM curd had a rigid protein matrix containing extensively cross-linked protein strands. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Importance of intrinsic properties of dense caseinate dispersions for structure formation.

PubMed

Manski, Julita M; van Riemsdijk, Lieke E; van der Goot, Atze J; Boom, Remko M

2007-11-01

Rheological measurements of dense calcium caseinate and sodium caseinate dispersions (> or =15%) provided insight into the factors determining shear-induced structure formation in caseinates. Calcium caseinate at a sufficiently high concentration (30%) was shown to form highly anisotropic structures during shearing and concurrent enzymatic cross-linking. In contrast, sodium caseinate formed isotropic structures using similar processing conditions. The main difference between the two types of caseinates is the counterion present, and as a consequence, the size of structural elements and their interactions. The rheological behavior of calcium caseinate and sodium caseinate reflected these differences, yielding non-monotonic and shear thinning flow behavior for calcium caseinate whereas sodium caseinate behaved only slightly shear thinning. It appears that the intrinsic properties of the dense caseinate dispersions, which are reflected in their rheological behavior, affect the structure formation that was found after applying shear. Therefore, rheological measurements are useful to obtain an indication of the structure formation potential of caseinate dispersions.

Rennet-induced coagulation properties of yak casein micelles: A comparison with cow casein micelles.

PubMed

Zhang, Yan; Li, Yuan; Wang, Pengjie; Tian, Yanbao; Liang, Qi; Ren, Fazheng

2017-12-01

It is essential for yak cheese processing to understand the rennet-induced coagulation properties of gel formation from casein micelles. We have previously discovered that yak milk requires a longer incubation time but forms stronger gels compared with cow milk. In this study, we are aiming to understand the rennet-induced coagulation properties of yak casein micelles comparing with cow casein micelles. Rheological analyses revealed that the gelling times of yak and cow casein micelles were 11.6±0.5 and 8.7±0.4min (P<0.05) respectively, but yak casein gel had a higher elastic modulus G' (6.5±0.2Pa) than cow casein gel (2.5±0.2Pa; P<0.05). This is consistent with the results obtained by micro-rheology. Confocal laser scanning microscopic images (CLSM) and cryo-scanning electron microscopic images (cryo-SEM) showed that yak casein gel was more homogeneous and had smaller pore size than cow casein gels. Yak casein micelles had higher calcium (26.00mM), phosphate (19.90mM) and β-casein (relative 32%) concentrations. In addition, yak casein micelles were larger (Z-average 218.6nm) than cow casein micelles, and contained lower κ-casein (relative 13%). By comparison with cow casein micelles, yak casein micelle composition corresponding to their micellar calcium phosphate and κ-casein content may greatly contribute to the longer coagulation time and denser gel structure. An initial slower caseinomacropeptide (CMP) release rate and the slower rate of aggregation between para-casein micelles contributed to a more homogeneous yak gel network. Higher colloidal calcium phosphate is crucial for yak casein micelle aggregation and gel firmness because sufficient colloidal calcium phosphates can firmly glue sub-micelles and links casein micelles. This study provides valuable information for yak cheese production. Copyright © 2017. Published by Elsevier Ltd.

αS1-casein, which is essential for efficient ER-to-Golgi casein transport, is also present in a tightly membrane-associated form

PubMed Central

2010-01-01

Background Caseins, the main milk proteins, aggregate in the secretory pathway of mammary epithelial cells into large supramolecular structures, casein micelles. The role of individual caseins in this process and the mesostructure of the casein micelle are poorly known. Results In this study, we investigate primary steps of casein micelle formation in rough endoplasmic reticulum-derived vesicles prepared from rat or goat mammary tissues. The majority of both αS1- and β-casein which are cysteine-containing casein was dimeric in the endoplasmic reticulum. Saponin permeabilisation of microsomal membranes in physico-chemical conditions believed to conserve casein interactions demonstrated that rat immature β-casein is weakly aggregated in the endoplasmic reticulum. In striking contrast, a large proportion of immature αS1-casein was recovered in permeabilised microsomes when incubated in conservative conditions. Furthermore, a substantial amount of αS1-casein remained associated with microsomal or post-ER membranes after saponin permeabilisation in non-conservative conditions or carbonate extraction at pH11, all in the presence of DTT. Finally, we show that protein dimerisation via disulfide bond is involved in the interaction of αS1-casein with membranes. Conclusions These experiments reveal for the first time the existence of a membrane-associated form of αS1-casein in the endoplasmic reticulum and in more distal compartments of the secretory pathway of mammary epithelial cells. Our data suggest that αS1-casein, which is required for efficient export of the other caseins from the endoplasmic reticulum, plays a key role in early steps of casein micelle biogenesis and casein transport in the secretory pathway. PMID:20704729

Development of casein microgels from cross-linking of casein micelles by genipin.

PubMed

Silva, Naaman F Nogueira; Saint-Jalmes, Arnaud; de Carvalho, Antônio F; Gaucheron, Frédéric

2014-09-02

Casein micelles are porous colloidal particles, constituted of casein molecules, water, and minerals. The vulnerability of the supramolecular structure of casein micelles face to changes in the environmental conditions restrains their applications in other domains besides food. Thus, redesigning casein micelles is a challenge to create new functionalities for these biosourced particles. The objective of this work was to create stable casein microgels from casein micelles using a natural cross-linker, named genipin. Suspensions of purified casein micelles (25 g L(-1)) were mixed with genipin solutions to have final concentrations of 5, 10, and 20 mM genipin. Covalently linked casein microgels were formed via cross-linking of lysyl and arginyl residues of casein molecules. The reacted products exhibited blue color. The cross-linking reaction induced gradual changes on the colloidal properties of the particles. The casein microgels were smaller and more negatively charged and presented smoother surfaces than casein micelles. These results were explained based on the cross-linking of free NH2 present in an external layer of κ-casein. Light scattering and rheological measurements showed that the reaction between genipin and casein molecules was intramicellar, as one single population of particles was observed and the values of viscosity (and, consequently, the volume fraction of the particles) were reduced. Contrary to the casein micelles, the casein microgels were resistant to the presence of dissociating agents, e.g., citrate (calcium chelating) and urea, but swelled as a consequence of internal electrostatic repulsion and the disruption of hydrophobic interactions between protein chains. The casein microgels did not dissociate at the air-solution interface and formed solid-like interfaces rather than a viscoelastic gel. The potential use of casein microgels as adaptable nanocarriers is proposed in the article.

Localization of the Netherton Syndrome Gene to Chromosome 5q32, by Linkage Analysis and Homozygosity Mapping

PubMed Central

Chavanas, Stéphane; Garner, Chad; Bodemer, Christine; Ali, Mohsin; Teillac, Dominique Hamel-; Wilkinson, John; Bonafé, Jean-Louis; Paradisi, Mauro; Kelsell, David P.; Ansai, Shin-ichi; Mitsuhashi, Yoshihiko; Larrègue, Marc; Leigh, Irene M.; Harper, John I.; Taïeb, Alain; Prost, Yves de; Cardon, Lon R.; Hovnanian, Alain

2000-01-01

Netherton syndrome (NS [MIM 256500]) is a rare and severe autosomal recessive disorder characterized by congenital ichthyosis, a specific hair-shaft defect (trichorrhexis invaginata), and atopic manifestations. Infants with this syndrome often fail to thrive; life-threatening complications result in high postnatal mortality. We report the assignment of the NS gene to chromosome 5q32, by linkage analysis and homozygosity mapping in 20 families affected with NS. Significant evidence for linkage (maximum multipoint LOD score 10.11) between markers D5S2017 and D5S413 was obtained, with no evidence for locus heterogeneity. Analysis of critical recombinants mapped the NS locus between markers D5S463 and D5S2013, within an <3.5-cM genetic interval. The NS locus is telomeric to the cytokine gene cluster in 5q31. The five known genes encoding casein kinase Iα, the α subunit of retinal rod cGMP phosphodiesterase, the regulator of mitotic-spindle assembly, adrenergic receptor β2, and the diastrophic dysplasia sulfate–transporter gene, as well as the 38 expressed-sequence tags mapped within the critical region, are not obvious candidates. Our study is the first step toward the positional cloning of the NS gene. This finding promises a better understanding of the molecular mechanisms that control epidermal differentiation and immunity. PMID:10712206

The chaperone action of bovine milk αS1- and αS2-caseins and their associated form αS-casein.

PubMed

Treweek, Teresa M; Thorn, David C; Price, William E; Carver, John A

2011-06-01

α(S)-Casein, the major milk protein, comprises α(S1)- and α(S2)-casein and acts as a molecular chaperone, stabilizing an array of stressed target proteins against precipitation. Here, we report that α(S)-casein acts in a similar manner to the unrelated small heat-shock proteins (sHsps) and clusterin in that it does not preserve the activity of stressed target enzymes. However, in contrast to sHsps and clusterin, α-casein does not bind target proteins in a state that facilitates refolding by Hsp70. α(S)-Casein was also separated into α- and α-casein, and the chaperone abilities of each of these proteins were assessed with amorphously aggregating and fibril-forming target proteins. Under reduction stress, all α-casein species exhibited similar chaperone ability, whereas under heat stress, α-casein was a poorer chaperone. Conversely, α(S2)-casein was less effective at preventing fibril formation by modified κ-casein, whereas α- and α(S1)-casein were comparably potent inhibitors. In the presence of added salt and heat stress, α(S1)-, α- and α(S)-casein were all significantly less effective. We conclude that α(S1)- and α-casein stabilise each other to facilitate optimal chaperone activity of α(S)-casein. This work highlights the interdependency of casein proteins for their structural stability. Copyright © 2011 Elsevier Inc. All rights reserved.

Absolute Quantification of Human Milk Caseins and the Whey/Casein Ratio during the First Year of Lactation.

PubMed

Liao, Yalin; Weber, Darren; Xu, Wei; Durbin-Johnson, Blythe P; Phinney, Brett S; Lönnerdal, Bo

2017-11-03

Whey proteins and caseins in breast milk provide bioactivities and also have different amino acid composition. Accurate determination of these two major protein classes provides a better understanding of human milk composition and function, and further aids in developing improved infant formulas based on bovine whey proteins and caseins. In this study, we implemented a LC-MS/MS quantitative analysis based on iBAQ label-free quantitation, to estimate absolute concentrations of α-casein, β-casein, and κ-casein in human milk samples (n = 88) collected between day 1 and day 360 postpartum. Total protein concentration ranged from 2.03 to 17.52 with a mean of 9.37 ± 3.65 g/L. Casein subunits ranged from 0.04 to 1.68 g/L (α-), 0.04 to 4.42 g/L (β-), and 0.10 to 1.72 g/L (α-), with β-casein having the highest average concentration among the three subunits. Calculated whey/casein ratio ranged from 45:55 to 97:3. Linear regression analyses show significant decreases in total protein, β-casein, κ-casein, total casein, and a significant increase of whey/casein ratio during the course of lactation. Our study presents a novel and accurate quantitative analysis of human milk casein content, demonstrating a lower casein content than earlier believed, which has implications for improved infants formulas.

Role of casein kinase 1A1 in the biology and targeted therapy of del(5q) MDS

PubMed Central

Schneider, Rebekka K.; Ademà, Vera; Heckl, Dirk; Järås, Marcus; Mallo, Mar; Lord, Allegra M.; Chu, Lisa P.; McConkey, Marie E.; Kramann, Rafael; Mullally, Ann; Bejar, Rafael; Solé, Francesc; Ebert, Benjamin L.

2014-01-01

Summary The Casein kinase 1A1 gene (CSNK1A1) is a putative tumor suppressor gene located in the common deleted region for del(5q) myelodysplastic syndrome (MDS). We generated a murine model with conditional inactivation of Csnk1a1 and found that Csnk1a1 haploinsufficiency induces hematopoietic stem cell expansion and a competitive repopulation advantage whereas homozygous deletion induces hematopoietic stem cell failure. Based on this finding, we found that heterozygous inactivation of Csnk1a1 sensitizes cells to a CSNK1 inhibitor relative to cells with two intact alleles. In addition, we identified recurrent somatic mutations in CSNK1A1 on the non-deleted allele of patients with del(5q) MDS. These studies demonstrate that CSNK1A1 plays a central role in the biology of del(5q) MDS and is a promising therapeutic target. PMID:25242043

Sequence polymorphisms at the growth hormone GH1/GH2-N and GH2-Z gene copies and their relationship with dairy traits in domestic sheep (Ovis aries).

PubMed

Vacca, G M; Dettori, M L; Balia, F; Luridiana, S; Mura, M C; Carcangiu, V; Pazzola, M

2013-09-01

The purpose was to analyze the growth hormone GH1/GH2-N and GH2-Z gene copies and to assess their possible association with milk traits in Sarda sheep. Two hundred multiparous lactating ewes were monitored. The two gene copies were amplified separately and each was used as template for a nested PCR, to investigate single strand conformation polymorphism (SSCP) of the 5'UTR, exon-1, exon-5 and 3'UTR DNA regions. SSCP analysis revealed marked differences in the number of polymorphic patterns between the two genes. Sequencing revealed five nucleotide changes at the GH1/GH2-N gene. Five nucleotide changes occurred at the GH2-Z gene: one was located in exon-5 (c.556G > A) and resulted in a putative amino acid substitution G186S. All the nucleotide changes were copy-specific, except c.*30delT, which was common to both GH1/GH2-N and GH2-Z. Variability in the promoter regions of each gene might have consequences on the expression level, due to the involvement in potential transcription factor binding sites. Both gene copies influenced milk yield. A correlation with milk protein and casein content was also evidenced. These results may have implications that make them useful for future breeding strategies in dairy sheep breeding.

Binding of vitamin A with milk α- and β-caseins.

PubMed

Bourassa, P; N'soukpoé-Kossi, C N; Tajmir-Riahi, H A

2013-05-01

The binding sites of retinol and retinoic acid with milk α- and β-caseins were determined, using constant protein concentration and various retinoid contents. FTIR, UV-visible and fluorescence spectroscopic methods as well as molecular modelling were used to analyse retinol and retinoic acid binding sites, the binding constant and the effect of retinoid complexation on the stability and conformation of caseins. Structural analysis showed that retinoids bind caseins via both hydrophilic and hydrophobic contacts with overall binding constants of K(retinol-)(α)(-caseins)=1.21 (±0.4)×10(5) M(-1) and K(retinol-)(β)(-caseins)=1.11 (±0.5)×10(5) M(-1) and K(retinoic acid-)(α)(-caseins)=6.2 (±0.6)×10(4) M(-1) and K(retinoic acid-)(β)(-caseins)=6.3 (±0.6)×10(4) M(-1). The number of bound retinol molecules per protein (n) was 1.5 (±0.1) for α-casein and 1.0 (±0.1) for β-casein, while 1 molecule of retinoic acid was bound in the α- and β-casein complexes. Molecular modelling showed different binding sites for retinol and retinoic acid on α- and β-caseins with more stable complexes formed with α-casein. Retinoid-casein complexation induced minor alterations of protein conformation. Caseins might act as carriers for transportation of retinoids to target molecules. Copyright © 2012 Elsevier Ltd. All rights reserved.

Supramolecular structure of the casein micelle.

PubMed

McMahon, D J; Oommen, B S

2008-05-01

The supramolecular structure of colloidal casein micelles in milk was investigated by using a sample preparation protocol based on adsorption of proteins onto a poly-l-lysine and parlodion-coated copper grid, staining of proteins and calcium phosphate by uranyl oxalate, instantaneous freezing, and drying under a high vacuum. High-resolution transmission electron microscopy stereo-images were obtained showing the interior structure of casein micelles. On the basis of our interpretation of these images, an interlocked lattice model was developed in which both casein-calcium phosphate aggregates and casein polymer chains act together to maintain casein micelle integrity. The caseins form linear and branched chains (2 to 5 proteins long) interlocked by the casein-stabilized calcium phosphate nanoclusters. This model suggests that stabilization of calcium phosphate nanoclusters by phosphoserine domains of alpha(s1)-, alpha(s2)-, or beta-casein, or their combination, would orient their hydrophobic domains outward, allowing interaction and binding to other casein molecules. Other interactions between the caseins, such as calcium bridging, could also occur and further stabilize the supramolecule. The combination of having an interlocked lattice structure and multiple interactions results in an open, sponge-like colloidal supramolecule that is resistant to spatial changes and disintegration. Hydrophobic interactions between caseins surrounding a calcium phosphate nanocluster would prevent complete dissociation of casein micelles when the calcium phosphate nanoclusters are solubilized. Likewise, calcium bridging and other electrostatic interactions between caseins would prevent dissociation of the casein micelles into casein-calcium phosphate nanocluster aggregates when milk is cooled or urea is added to milk, and hydrophobic interactions are reduced. The appearance of both polymer chains and small aggregate particles during milk synthesis would also be expected based on this interlocked lattice model of casein micelles, and its supramolecule structure thus exhibits the principles of self-aggregation, interdependence, and diversity observed in nature.

Production of calcium- and magnesium-enriched caseins and caseinates by an ecofriendly technology.

PubMed

Masson, Félix-André; Mikhaylin, Sergey; Bazinet, Laurent

2018-05-09

Finding new green ways of producing proteins has never been of such critical public interest, both to meet consumers' needs and to preserve the environment. Milk proteins are among the most attractive protein types due to their high nutritional value and attractive functional properties. In this work, the separation of caseins by conventional chemical acidification was compared with electrodialysis with bipolar membrane coupled to an ultrafiltration module (EDBM-UF), a green process that allows the precipitation of caseins by H + generated in situ by the bipolar membrane and, simultaneously, the production of a separated NaOH stream from OH - electrogenerated by the bipolar membrane. Caseinate production using this NaOH stream by-product and the quantity of NaOH needed to produce caseinates from both methods were also investigated. Hence, the purity and composition of caseins and caseinates were compared in terms of protein, ash, and lactose contents as well as mineral composition. The results showed for the first time that caseinates can be produced by solubilizing caseins with NaOH stream from the EDBM process. Furthermore, the caseins and caseinates produced by EDBM-UF were equivalent in terms of lactose and protein contents to their respective caseins and caseinates that were chemically produced but presented slightly lower sodium content and 3 to 4 times higher magnesium and calcium contents. The fact that calcium and magnesium are likely bound to milk caseins would ensure their favorable absorbability. These caseins or caseinates from the new EDBM-UF process could be suitable as an improved protein-based calcium or magnesium supplement, both for their enhanced nutritional quality and because they are produced by a green process. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Antimicrobial activity and mechanism of the human milk-sourced peptide Casein201

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Fan; Department of Endocrinology, Children's Hospital of Nanjing Medical University, Nanjing; Cui, Xianwei

Introduction: Casein201 is one of the human milk sourced peptides that differed significantly in preterm and full-term mothers. This study is designed to demonstrate the biological characteristics, antibacterial activity and mechanisms of Casein201 against common pathogens in neonatal infection. Methodology: The analysis of biological characteristics was done by bioinformatics. Disk diffusion method and flow cytometry were used to detect the antimicrobial activity of Casein201. Killing kinetics of Casein201 was measured using microplate reader. The antimicrobial mechanism of Casein201 was studied by electron microscopy and electrophoresis. Results: Bioinformatics analysis indicates that Casein201 derived from β-casein and showed significant sequence overlap. Antibacterialmore » assays showed Casein201 inhibited the growth of S taphylococcus aureus and Y ersinia enterocolitica. Ultrastructural analyses revealed that the antibacterial activity of Casein201 is through cytoplasmic structures disintegration and bacterial cell envelope alterations but not combination with DNA. Conclusion: We conclude the antimicrobial activity and mechanism of Casein201. Our data demonstrate that Casein201 has potential therapeutic value for the prevention and treatment of pathogens in neonatal infection.« less

Antimicrobial activity and mechanism of the human milk-sourced peptide Casein201.

PubMed

Zhang, Fan; Cui, Xianwei; Fu, Yanrong; Zhang, Jun; Zhou, Yahui; Sun, Yazhou; Wang, Xing; Li, Yun; Liu, Qianqi; Chen, Ting

2017-04-08

Casein201 is one of the human milk sourced peptides that differed significantly in preterm and full-term mothers. This study is designed to demonstrate the biological characteristics, antibacterial activity and mechanisms of Casein201 against common pathogens in neonatal infection. The analysis of biological characteristics was done by bioinformatics. Disk diffusion method and flow cytometry were used to detect the antimicrobial activity of Casein201. Killing kinetics of Casein201 was measured using microplate reader. The antimicrobial mechanism of Casein201 was studied by electron microscopy and electrophoresis. Bioinformatics analysis indicates that Casein201 derived from β-casein and showed significant sequence overlap. Antibacterial assays showed Casein201 inhibited the growth of S taphylococcus aureus and Y ersinia enterocolitica. Ultrastructural analyses revealed that the antibacterial activity of Casein201 is through cytoplasmic structures disintegration and bacterial cell envelope alterations but not combination with DNA. We conclude the antimicrobial activity and mechanism of Casein201. Our data demonstrate that Casein201 has potential therapeutic value for the prevention and treatment of pathogens in neonatal infection. Copyright © 2017 Elsevier Inc. All rights reserved.

Iron binding to caseins in the presence of orthophosphate.

PubMed

Mittal, V A; Ellis, A; Ye, A; Edwards, P J B; Das, S; Singh, H

2016-01-01

As adding >5mM ferric chloride to sodium caseinate solutions results in protein precipitation, the effects of orthophosphate (0-64 mM) addition to sodium caseinate solution (2% w/v protein) on iron-induced aggregation of the caseins were studied at pH 6.8. Up to 20mM ferric chloride could be added to sodium caseinate solution containing 32 mM orthophosphate without any protein precipitation. The addition of iron to sodium caseinate solution containing orthophosphate reduced the diffusible phosphorus content in a concentration-dependent manner. Added iron appeared to interact simultaneously with phosphoserine on the caseins and inorganic phosphorus. The relative sizes of the casein aggregates were governed by the concentration of orthophosphate and the aggregates consisted of all casein fractions, even at the lowest level of ferric chloride addition (5mM). It is hypothesised that the addition of iron to caseins in the presence of orthophosphate results in the formation of colloidal structures involving casein-iron-orthophosphate interactions. Copyright © 2015 Elsevier Ltd. All rights reserved.

Electrochemical Sensing of Casein Based on the Interaction between Its Phosphate Groups and a Ruthenium(III) Complex.

PubMed

Inaba, Iku; Kuramitz, Hideki; Sugawara, Kazuharu

2016-01-01

A reaction to casein, along with β-lactoglobulin, is a main cause of milk allergies, and also is a useful indicator of protein in allergic analyses. In the present study, a simple casein sensor was developed based on the interaction between a phosphate group of casein and electroactive [Ru(NH3)6](3+). We evaluated the voltammetric behavior of a casein-[Ru(NH3)6](3+) complex using a glassy carbon electrode. When the ruthenium(III) complex was combined with the phosphate groups of casein, the structure of the casein was changed. Since the hydrophobicity of casein was increased due to the binding, the casein was adsorbed onto the electrode. Furthermore, we modified an electrode with a ruthenium(III) ions/collagen film. When the sensor was applied to the detection of the casein contained in milk, the values coincided with those indicated by the manufacturer. Accordingly, this electrode could be a powerful sensor for the determination of casein in several foods.

Proteomic analysis to unravel the effect of heat stress on gene expression and milk synthesis in bovine mammary epithelial cells.

PubMed

Li, Lian; Wang, Yiru; Li, Chengmin; Wang, Genlin

2017-12-01

Heat stress can play a negative effect on milk yield and composition of dairy cattle, leading to immeasurable economic loss. The basic components of the mammary gland are the alveoli; these alveolar mammary epithelial cells reflect the milk producing ability of dairy cows. In this study, we exposed bovine mammary epithelial cells to heat stress and compared them to a control group using isobaric tags for relative and absolute quantitation combined with liquid chromatography coupled with tandem mass spectrometry. Compared with a control group, 104 differentially elevated proteins (>1.3-fold) and 167 decreased proteins (<0.77-fold) were identified in the heat treatment group. Gene Ontology analysis identified a majority of the differentially expressed proteins are associated in cell-substrate junction assembly, catabolic processes and metabolic processes. Some of these significantly regulated proteins were related to the synthesis and secretion of milk, such as milk protein and fat. This finding was further supported by the results obtained from the reduced β-casein expression through the system of plasminogen activator - plasminogen - plasmin and decreased fatty acid synthase could partly explain why milk fat synthesis ability of dairy cows decreased under heat stress. Our results highlight the effects of heat stress on synthesis of milk protein and fat, thus providing additional clues for further studies of heat stress on dairy milk production. © 2017 Japanese Society of Animal Science.

Metabolomics Reveals Aryl Hydrocarbon Receptor Activation Induces Liver and Mammary Gland Metabolic Dysfunction in Lactating Mice.

PubMed

Belton, Kerry R; Tian, Yuan; Zhang, Limin; Anitha, Mallappa; Smith, Philip B; Perdew, Gary H; Patterson, Andrew D

2018-04-06

The liver and the mammary gland have complementary metabolic roles during lactation. Substrates synthesized by the liver are released into the circulation and are taken up by the mammary gland for milk production. The aryl hydrocarbon receptor (AHR) has been identified as a lactation regulator in mice, and its activation has been associated with myriad morphological, molecular, and functional defects such as stunted gland development, decreased milk production, and changes in gene expression. In this study, we identified adverse metabolic changes in the lactation network (mammary, liver, and serum) associated with AHR activation using 1 H nuclear magnetic resonance (NMR)-based metabolomics. Pregnant mice expressing Ahr d (low affinity) or Ahr b (high affinity) were fed diets containing beta naphthoflavone (BNF), a potent AHR agonist. Mammary, serum, and liver metabolomics analysis identified significant changes in lipid and TCA cycle intermediates in the Ahr b mice. We observed decreased amino acid and glucose levels in the mammary gland extracts of Ahr b mice fed BNF. The serum of BNF fed Ahr b mice had significant changes in LDL/VLDL (increased) and HDL, PC, and GPC (decreased). Quantitative PCR analysis revealed ∼50% reduction in the expression of key lactogenesis mammary genes including whey acid protein, α-lactalbumin, and β-casein. We also observed morphologic and developmental disruptions in the mammary gland that are consistent with previous reports. Our observations support that AHR activity contributes to metabolism regulation in the lactation network.

Interactions of casein micelles with calcium phosphate particles.

PubMed

Tercinier, Lucile; Ye, Aiqian; Anema, Skelte G; Singh, Anne; Singh, Harjinder

2014-06-25

Insoluble calcium phosphate particles, such as hydroxyapatite (HA), are often used in calcium-fortified milks as they are considered to be chemically unreactive. However, this study showed that there was an interaction between the casein micelles in milk and HA particles. The caseins in milk were shown to bind to the HA particles, with the relative proportions of bound β-casein, αS-casein, and κ-casein different from the proportions of the individual caseins present in milk. Transmission electron microscopy showed no evidence of intact casein micelles on the surface of the HA particles, which suggested that the casein micelles dissociated either before or during binding. The HA particles behaved as ion chelators, with the ability to bind the ions contained in the milk serum phase. Consequently, the depletion of the serum minerals disrupted the milk mineral equilibrium, resulting in dissociation of the casein micelles in milk.

Expression and characterization of camel chymosin in Pichia pastoris.

PubMed

Wang, Nan; Wang, Kevin Yueju; Li, GangQiang; Guo, WenFang; Liu, DeHu

2015-07-01

Chymosin efficiently coagulates milk and so is widely used in commercial cheese production. Traditional chymosin production requires the slaughter of a large numbers of unweaned calves. In the present study, a full-length camel prochymosin gene was synthesized and cloned into the pPIC9K vector, which was then inserted into the yeast strain, Pichia pastoris GS115. Expression of the chymosin gene in yeast was under the control of an AOX1 inducible promoter. The yeast system produced approximately 37mg/L of recombinant enzyme under lab conditions. SDS-PAGE of the raw supernatant revealed two molecular bands, which were approximately 42kDa and 45kDa in size. The 45kDa band disappeared after treatment of the supernatant with N-glycosidase F (PNGase F), indicating that the recombinant protein was partially glycosylated. When subjected to a low pH, recombinant prochymosin was converted into mature and active chymosin. The active chymosin was capable of specifically hydrolyzing κ-casein. A pH of 5.04, and temperature range of 45-50°C, was optimum for milk clotting activity. Maximum milk clotting activity was detected with the inclusion of 20-40mM CaCl2. The recombinant enzyme was highly active and stable over a wide pH range (from 2.5 to 6.5) at 20°C for 8h. Thermostability of the recombinant enzyme was also analyzed. Pilot-scale production (300mg/L) was attained using a 5L fermenter. We demonstrated that expression of the camel chymosin gene in P. pastoris could represent an excellent system for producing active camel chymosin for potential use in the commercial production of cheese. Copyright © 2015 Elsevier Inc. All rights reserved.

STUDIES ON THE FORMATION AND IONIZATION OF THE COMPOUNDS OF CASEIN WITH ALKALI

PubMed Central

Greenberg, David M.; Schmidt, Carl L. A.

1924-01-01

1. The deposition of casein on a platinum anode which takes place on the passage of a direct current through solutions of alkali caseinates was quantitatively studied, and it was found that: (a) the amount of casein which is deposited is directly proportional to the current, i.e. it obeys Faraday's law; (b) the amount of casein deposited is inversely proportional (within the limits studied) to the amount of alkali which is combined with the casein. 2. A method of determining the transport numbers of proteins insoluble at their isoelectric point has been developed. 3. A titration method for determining the amount of alkali in a casein solution is given. 4. Data from the results of transference experiments on sodium caseinate, potassium caseinate, cesium caseinate, and rubidium caseinate solutions are given. It is shown that the data are best explained on the assumption that in these solutions the carriers of the current are alkali metal cations and casein anions. 5. On the basis of our transference results an explanation is given of the results which were obtained by Robertson and by Haas in their migration experiments. PMID:19872135

Complexes of lutein with bovine and caprine caseins and their impact on lutein chemical stability in emulsion systems: Effect of arabinogalactan.

PubMed

Mora-Gutierrez, A; Attaie, R; Núñez de González, M T; Jung, Y; Woldesenbet, S; Marquez, S A

2018-01-01

Lutein is an important xanthophyll carotenoid with many benefits to human health. Factors affecting the application of lutein as a functional ingredient in low-fat dairy-like beverages (pH 6.0-7.0) are not well understood. The interactions of bovine and caprine caseins with hydrophobic lutein were studied using UV/visible spectroscopy as well as fluorescence. Our studies confirmed that the aqueous solubility of lutein is improved after binding with bovine and caprine caseins. The rates of lutein solubilization by the binding to bovine and caprine caseins were as follows: caprine α S1 -II-casein 34%, caprine α S1 -I-casein 10%, and bovine casein 7% at 100 μM lutein. Fluorescence of the protein was quenched on binding supporting complex formation. The fluorescence experiments showed that the binding involves tryptophan residues and some nonspecific interactions. Scatchard plots of lutein binding to the caseins demonstrated competitive binding between the caseins and their sites of interaction with lutein. Competition experiments suggest that caprine α S1 -II casein will bind a larger number of lutein molecules with higher affinity than other caseins. The chemical stability of lutein was largely dependent on casein type and significant increases occurred in the chemical stability of lutein with the following pattern: caprine α S1 -II-casein > caprine α S1 -I-casein > bovine casein. Addition of arabinogalactan to lutein-enriched emulsions increases the chemical stability of lutein-casein complexes during storage under accelerated photo-oxidation conditions at 25°C. Therefore, caprine α S1 -II-casein alone and in combination with arabinogalactan can have important applications in the beverage industry as carrier of this xanthophyll carotenoid (lutein). Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Casein Aggregates Built Step-by-Step on Charged Polyelectrolyte Film Surfaces Are Calcium Phosphate-cemented*

PubMed Central

Nagy, Krisztina; Pilbat, Ana-Maria; Groma, Géza; Szalontai, Balázs; Cuisinier, Frédéric J. G.

2010-01-01

The possible mechanism of casein aggregation and micelle buildup was studied in a new approach by letting α-casein adsorb from low concentration (0.1 mg·ml−1) solutions onto the charged surfaces of polyelectrolyte films. It was found that α-casein could adsorb onto both positively and negatively charged surfaces. However, only when its negative phosphoseryl clusters remained free, i.e. when it adsorbed onto a negative surface, could calcium phosphate (CaP) nanoclusters bind to the casein molecules. Once the CaP clusters were in place, step-by-step building of multilayered casein architectures became possible. The presence of CaP was essential; neither Ca2+ nor phosphate could alone facilitate casein aggregation. Thus, it seems that CaP is the organizing motive in the casein micelle formation. Atomic force microscopy revealed that even a single adsorbed casein layer was composed of very small (in the range of tens of nanometers) spherical forms. The stiffness of the adsorbed casein layer largely increased in the presence of CaP. On this basis, we can imagine that casein micelles emerge according to the following scheme. The amphipathic casein monomers aggregate into oligomers via hydrophobic interactions even in the absence of CaP. Full scale, CaP-carrying micelles could materialize by interlocking these casein oligomers with CaP nanoclusters. Such a mechanism would not contradict former experimental results and could offer a synthesis between the submicelle and the block copolymer models of casein micelles. PMID:20921229

Locating the binding sites of folic acid with milk α- and β-caseins.

PubMed

Bourassa, P; Tajmir-Riahi, H A

2012-01-12

We located the binding sites of folic acid with milk α- and β-caseins at physiological conditions, using constant protein concentration and various folic acid contents. FTIR, UV-visible, and fluorescence spectroscopic methods as well as molecular modeling were used to analyze folic acid binding sites, the binding constant, and the effect of folic acid interaction on the stability and conformation of caseins. Structural analysis showed that folic acid binds caseins via both hydrophilic and hydrophobic contacts with overall binding constants of K(folic acid-α-caseins) = 4.8 (±0.6) × 10(4) M(-1) and K(folic acid-β-caseins) = 7.0 (±0.9) × 10(4) M(-1). The number of bound acid molecules per protein was 1.5 (±0.4) for α-casein and 1.4 (±0.3) for β-casein complexes. Molecular modeling showed different binding sites for folic acid on α- and β-caseins. The participation of several amino acids in folic acid-protein complexes was observed, which was stabilized by hydrogen bonding network and the free binding energy of -7.7 kcal/mol (acid-α-casein) and -8.1 kcal/mol (acid-β-casein). Folic acid complexation altered protein secondary structure by the reduction of α-helix from 35% (free α-casein) to 33% (acid-complex) and 32% (free β-casein) to 26% (acid-complex) indicating a partial protein destabilization. Caseins might act as carriers for transportation of folic acid to target molecules.

The dominant expression of functional human lactoferrin in transgenic cloned goats using a hybrid lactoferrin expression construct.

PubMed

Yu, Huiqing; Chen, Jianquan; Sun, Wei; Liu, Siguo; Zhang, Aimin; Xu, Xujun; Wang, Xuebin; He, Zhuzi; Liu, Guohui; Cheng, Guoxiang

2012-10-31

Human Lactoferrin (hLF) is an iron-binding protein with multiple physiological functions. As the availability of natural hLF is limited, alternative means of producing this biopharmaceutical protein have been extensively studied. Here we report on the dominant expression of recombinant human lactoferrin (rhLF) in transgenic cloned goats using a novel optimised construct made by fusing a 3.3 kb hLF minigene to the regulatory elements of the β-casein gene. The transgenic goat produced more than 30 mg/ml rhLF in its milk, and rhLF expression was stable during the entire lactation cycle. The rhLF purification efficiency from whole goat milk is approximately 70%, and its purity is above 98%. Compared with natural hLF, the rhLF from transgenic goats has similar biological characteristics including molecular mass, N-terminal sequence, isoelectric point, immunoreactivity and digestive stability. More importantly, the purified rhLF showed specific anti-tumour activity in the mouse model of melanoma experimental metastasis. Therefore, our study shows that the large-scale production of functional rhLF in transgenic goat milk could be an economical and promising source of human therapeutic use in the future. Copyright © 2012. Published by Elsevier B.V.

Drosophila Casein Kinase I Alpha Regulates Homolog Pairing and Genome Organization by Modulating Condensin II Subunit Cap-H2 Levels

PubMed Central

Nguyen, Huy Q.; Nye, Jonathan; Buster, Daniel W.; Klebba, Joseph E.; Rogers, Gregory C.; Bosco, Giovanni

2015-01-01

The spatial organization of chromosomes within interphase nuclei is important for gene expression and epigenetic inheritance. Although the extent of physical interaction between chromosomes and their degree of compaction varies during development and between different cell-types, it is unclear how regulation of chromosome interactions and compaction relate to spatial organization of genomes. Drosophila is an excellent model system for studying chromosomal interactions including homolog pairing. Recent work has shown that condensin II governs both interphase chromosome compaction and homolog pairing and condensin II activity is controlled by the turnover of its regulatory subunit Cap-H2. Specifically, Cap-H2 is a target of the SCFSlimb E3 ubiquitin-ligase which down-regulates Cap-H2 in order to maintain homologous chromosome pairing, chromosome length and proper nuclear organization. Here, we identify Casein Kinase I alpha (CK1α) as an additional negative-regulator of Cap-H2. CK1α-depletion stabilizes Cap-H2 protein and results in an accumulation of Cap-H2 on chromosomes. Similar to Slimb mutation, CK1α depletion in cultured cells, larval salivary gland, and nurse cells results in several condensin II-dependent phenotypes including dispersal of centromeres, interphase chromosome compaction, and chromosome unpairing. Moreover, CK1α loss-of-function mutations dominantly suppress condensin II mutant phenotypes in vivo. Thus, CK1α facilitates Cap-H2 destruction and modulates nuclear organization by attenuating chromatin localized Cap-H2 protein. PMID:25723539

Casein kinase 1 regulates sterol regulatory element-binding protein (SREBP) to control sterol homeostasis.

PubMed

Brookheart, Rita T; Lee, Chih-Yung S; Espenshade, Peter J

2014-01-31

Sterol homeostasis is tightly controlled by the sterol regulatory element-binding protein (SREBP) transcription factor that is highly conserved from fungi to mammals. In fission yeast, SREBP functions in an oxygen-sensing pathway to promote adaptation to decreased oxygen supply that limits oxygen-dependent sterol synthesis. Low oxygen stimulates proteolytic cleavage of the SREBP homolog Sre1, generating the active transcription factor Sre1N that drives expression of sterol biosynthetic enzymes. In addition, low oxygen increases the stability and DNA binding activity of Sre1N. To identify additional signals controlling Sre1 activity, we conducted a genetic overexpression screen. Here, we describe our isolation and characterization of the casein kinase 1 family member Hhp2 as a novel regulator of Sre1N. Deletion of Hhp2 increases Sre1N protein stability and ergosterol levels in the presence of oxygen. Hhp2-dependent Sre1N degradation by the proteasome requires Hhp2 kinase activity, and Hhp2 binds and phosphorylates Sre1N at specific residues. Our results describe a role for casein kinase 1 as a direct regulator of sterol homeostasis. Given the role of mammalian Hhp2 homologs, casein kinase 1δ and 1ε, in regulation of the circadian clock, these findings may provide a mechanism for coordinating circadian rhythm and lipid metabolism.

The bioactive effects of casein proteins on enteroendocrine cell health, proliferation and incretin hormone secretion.

PubMed

Gillespie, Anna L; Green, Brian D

2016-11-15

Previous studies suggest that casein exerts various anti-diabetic effects. However, it is not known which casein proteins are bioactive, nor their effects on enteroendocrine cells. This study evaluated the effects of intact whole casein, intact individual proteins (alpha, beta and kappa casein) and hydrolysates on an enteroendocrine cell line. High content analysis accurately monitored changes in cell health and intracellular glucagon-like peptide-1 (GLP-1) content. Cheese ripening duration and GLP-1 secretory responses were also considered. Beta casein significantly stimulated enteroendocrine cell proliferation and all caseins were potent GLP-1 secretagogues (except kappa casein). Interestingly the GLP-1 secretory activity was almost always lost or significantly reduced upon hydrolysis with proteolytic enzymes. Only pepsin-derived beta casein hydrolysates had significantly increased potency compared with the intact protein, but this was diminished with prolonged hydrolysis. In conclusion casein proteins are not detrimental to enteroendocrine cells, and alpha and beta casein are particularly beneficial stimulating proliferation and GLP-1 secretion. Copyright © 2016 Elsevier Ltd. All rights reserved.

Topography of the casein micelle surface by surface plasmon resonance (SPR) using a selection of specific monoclonal antibodies.

PubMed

Dupont, Didier; Johansson, Annette; Marchin, Stephane; Rolet-Repecaud, Odile; Marchesseau, Sylvie; Leonil, Joelle

2011-08-10

Several theoretical models of the casein micelle structure have been proposed in the past, but the exact organization of the four individual caseins (α(s1), α(s2), β, and κ) within this supramolecular structure remains unknown. The present study aims at determining the topography of the casein micelle surface by following the interaction between 44 monoclonal antibodies specific for different epitopes of α(s1)-, α(s2)-, β-, and κ-casein and the casein micelle in real time and no labeling using a surface plasmon resonance (SPR)-based biosensor. Although the four individual caseins were found to be accessible for antibody binding, data confirmed that the C-terminal extremity of κ-casein was highly accessible and located at the periphery of the structure. When casein micelles were submitted to proteolysis, the C-terminal extremity of κ-casein was rapidly hydrolyzed. Disintegration of the micellar structure resulted in an increased access for antibodies to hydrophobic areas of α(s1)- and α(s2)-casein.

Short communication: Effect of casein haplotype on angiotensin-converting enzyme inhibitory and antioxidant capacities of milk casein from Italian Holstein cows before and following in vitro digestion with gastrointestinal enzymes.

PubMed

Perna, Annamaria; Simonetti, Amalia; Gambacorta, Emilio

2016-09-01

The aim of this work was to investigate the effect of casein haplotype (αS1, β, and κ) on antioxidative and angiotensin-converting enzyme (ACE) inhibitory capacities of milk casein from Italian Holstein cows before and following in vitro digestion with gastrointestinal enzymes. The antioxidant capacity was measured using 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid and ferric-reducing antioxidant power assays, whereas ACE inhibition was determined by ACE-inhibitory assay. The ACE-inhibitory and antioxidant capacities of milk casein increased during in vitro gastrointestinal digestion. Casein haplotype significantly influenced the antioxidative and ACE-inhibitory capacities of digested casein. In particular, BB-A(2)A(1)-AA casein and BB-A(1)A(1)-AA casein showed the highest ACE-inhibitory capacity, BB-A(2)A(2)-AA casein showed the highest antioxidant capacity, whereas BB-A(2)A(2)-BB casein showed the lowest biological capacity. To date, few studies have been done on the effect of casein haplotype on biological capacity of milk casein, thus the present study sets the basis for a new knowledge that could lead to the production of milk with better nutraceutical properties. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Use of calcium caseinate in association with lecithin for masking the bitterness of acetaminophen--comparative study with sodium caseinate.

PubMed

Hoang Thi, Thanh Huong; Lemdani, Mohamed; Flament, Marie-Pierre

2013-11-18

Owing to a variety of structural and functional properties, milk proteins are steadily studied for food and pharmaceutical applications. In the present study, calcium caseinate in association with lecithin was firstly investigated in order to encapsulate the acetaminophen through spray-drying for taste-masking purpose for pediatric medicines. A 2(4)-full factorial design revealed that the spray flow, the calcium caseinate amount and the lecithin amount had significant effects on the release of drug during the first 2 min. Indeed, increasing the spray flow and/or the calcium caseinate amount led to increase the released amount, whereas increasing the lecithin amount decreased the released amount. The "interaction" between the calcium caseinate amount and the lecithin amount was also shown to be statistically significant. The second objective was to compare the efficiency of two caseinate-based formulations, i.e. sodium caseinate and calcium caseinate, on the taste-masking effect. The characteristics of spray-dried powders determined by SEM and DSC were shown to depend on the caseinate/lecithin proportion rather than the type of caseinate. Interestingly, calcium caseinate-based formulations were found to lower the released amount of drug during the early time to a higher extent than sodium caseinate-based formulations, which indicates better taste-masking efficiency. Copyright © 2013 Elsevier B.V. All rights reserved.

The adsorption of orthophosphate onto casein-iron precipitates.

PubMed

Mittal, Vikas A; Ellis, Ashling; Ye, Aiqian; Edwards, Patrick J B; Singh, Harjinder

2018-01-15

This study explored the interactions of orthophosphate with casein-iron precipitates. Casein-iron precipitates were formed by adding ferric chloride at ≥10mM to sodium caseinate solutions ranging in concentration from 1 to 3%(w/v). The addition of different concentrations of orthophosphate solution to the casein-iron precipitates resulted in gradual adsorption of the orthophosphate, causing re-dispersion of the casein-iron complexes. The interactions of added orthophosphate with iron in the presence and absence of caseins are postulated, and new mechanisms are proposed. The re-dispersed soluble complexes of casein-iron-orthophosphate generated using this process could be used as novel iron fortificants. Copyright © 2017 Elsevier Ltd. All rights reserved.

pH-dependent structures and properties of casein micelles.

PubMed

Liu, Yan; Guo, Rong

2008-08-01

The association behavior of casein over a broad pH range has first been investigated by fluorescent technique together with DLS and turbidity measurements. Casein molecules can self-assemble into casein micelles in the pH ranges 2.0 to 3.0, and 5.5 to 12.0. The hydrophobic interaction, hydrogen bond and electrostatic action are the main interactions in the formation of casein micelles. The results show that the structure of casein micelles is more compact at low pH and looser at high pH. The casein micelle has the most compact structure at pH 5.5, when it has almost no electrostatic repulsion between casein molecules.

Structural changes of casein micelles in a calcium gradient film.

PubMed

Gebhardt, Ronald; Burghammer, Manfred; Riekel, Christian; Roth, Stephan Volkher; Müller-Buschbaum, Peter

2008-04-09

Calcium gradients are prepared by sequentially filling a micropipette with casein solutions of varying calcium concentration and spreading them on glass slides. The casein film is formed by a solution casting process, which results in a macroscopically rough surface. Microbeam grazing incidence small-angle X-ray scattering (microGISAXS) is used to investigate the lateral size distribution of three main components in casein films: casein micelles, casein mini-micelles, and micellar calcium phosphate. At length scales within the beam size the film surface is flat and detection of size distribution in a macroscopic casein gradient becomes accessible. The model used to analyze the data is based on a set of three log-normal distributed particle sizes. Increasing calcium concentration causes a decrease in casein micelle diameter while the size of casein mini-micelles increases and micellar calcium phosphate particles remain unchanged.

THE ANTIGENIC PROPERTIES OF GLOBIN CASEINATE

PubMed Central

Gay, Frederick P.; Robertson, T. Brailsford

1913-01-01

This study of globin and its compound with casein (globin caseinate) shows that globin fails to produce fixation antibodies in rabbits after repeated injections, thus agreeing with our own work and with that of others with similar histon bodies which are primarily toxic. When globin is combined with casein, however, it gives rise to antibodies that react not only with globin caseinate and casein but also with globin. The antibodies in antiglobin casein serum are apparently separate, one for globin and one for casein. In other words, the change in globin undergone on combination with casein has apparently rendered it antigenic. We did not succeed in demonstrating the genesis of this new antigenic property by anaphylaxis experiments. A further investigation of similar and more complex combined proteins is indicated and gives promise of more light on the nature of biological specificity. PMID:19867665

Whey Protein Reduces Early Life Weight Gain in Mice Fed a High-Fat Diet

PubMed Central

Tranberg, Britt; Hellgren, Lars I.; Lykkesfeldt, Jens; Sejrsen, Kristen; Jeamet, Aymeric; Rune, Ida; Ellekilde, Merete; Nielsen, Dennis S.; Hansen, Axel Kornerup

2013-01-01

An increasing number of studies indicate that dairy products, including whey protein, alleviate several disorders of the metabolic syndrome. Here, we investigated the effects of whey protein isolate (whey) in mice fed a high-fat diet hypothesising that the metabolic effects of whey would be associated with changes in the gut microbiota composition. Five-week-old male C57BL/6 mice were fed a high-fat diet ad libitum for 14 weeks with the protein source being either whey or casein. Faeces were collected at week 0, 7, and 13 and the fecal microbiota was analysed by denaturing gradient gel electrophoresis analyses of PCR-derived 16S rRNA gene (V3-region) amplicons. At the end of the study, plasma samples were collected and assayed for glucose, insulin and lipids. Whey significantly reduced body weight gain during the first four weeks of the study compared with casein (P<0.001–0.05). Hereafter weight gain was similar resulting in a 15% lower final body weight in the whey group relative to casein (34.0±1.0 g vs. 40.2±1.3 g, P<0.001). Food intake was unaffected by protein source throughout the study period. Fasting insulin was lower in the whey group (P<0.01) and glucose clearance was improved after an oral glucose challenge (P<0.05). Plasma cholesterol was lowered by whey compared to casein (P<0.001). The composition of the fecal microbiota differed between high- and low-fat groups at 13 weeks (P<0.05) whereas no difference was seen between whey and casein. In conclusion, whey initially reduced weight gain in young C57BL/6 mice fed a high-fat diet compared to casein. Although the effect on weight gain ceased, whey alleviated glucose intolerance, improved insulin sensitivity and reduced plasma cholesterol. These findings could not be explained by changes in food intake or gut microbiota composition. Further studies are needed to clarify the mechanisms behind the metabolic effects of whey. PMID:23940754

Reformation of casein particles from alkaline-disrupted casein micelles.

PubMed

Huppertz, Thom; Vaia, Betsy; Smiddy, Mary A

2008-02-01

In this study, the properties of casein particles reformed from alkaline disrupted casein micelles were studied. For this purpose, micelles were disrupted completely by increasing milk pH to 10.0, and subsequently reformed by decreasing milk pH to 6.6. Reformed casein particles were smaller than native micelles and had a slightly lower zeta-potential. Levels of ionic and serum calcium, as well as rennet coagulation time did not differ between milk containing native micelles or reformed casein particles. Ethanol stability and heat stability, >pH 7.0, were lower for reformed casein particles than native micelles. Differences in heat stability, ethanol stability and zeta-potential can be explained in terms of the influence of increased concentrations of sodium and chloride ions in milk containing reformed casein particles. Hence, these results indicate that, if performed in a controlled manner, casein particles with properties closely similar to those of native micelles can be reformed from alkaline disrupted casein micelles.

The sheep growth hormone gene polymorphism and its effects on milk traits.

PubMed

Dettori, Maria Luisa; Pazzola, Michele; Pira, Emanuela; Paschino, Pietro; Vacca, Giuseppe Massimo

2015-05-01

Growth hormone (GH) is encoded by the GH gene, which may be single copy or duplicate in sheep. The two copies of the sheep GH gene (GH1/GH2-N and GH2-Z) were entirely sequenced in one 106 ewes of Sarda breed, in order to highlight sequence polymorphisms and investigate possible association between genetic variants and milk traits. Milk traits included milk yield, fat, protein, casein and lactose percentage. We evidenced 75 nucleotide changes. Transcription factor binding site prediction revealed two sequences potentially recognised by the pituitary-specific transcription factor POU1FI at the GH1/GH2-N gene, which were lost at the promoter of GH2-Z, which might explain the different tissues of expression of GH1/GH2-N (pituitary) and GH2-Z (placenta). Significant differences in milk traits were observed among genotypes at polymorphic loci only for the GH2-Z gene. Sheep with homozygote genotype ss748770547 CC had higher fat percentage (P < 0.01) than TT. SNP ss748770547 was part of a potential transcription factor binding site for C/EBP alpha (CCAAT/Enhancer Binding Protein), which is involved in the regulation of adipogenesis and adipoblast differentiation. SNP ss748770547, located in the GH2-Z gene 5' flanking region, may be a causal mutation affecting milk fat content. These findings might contribute to the knowledge of the sheep GH locus and might be useful in selection processes in sheep.

Branched-Chain Amino Acids Ameliorate Fibrosis and Suppress Tumor Growth in a Rat Model of Hepatocellular Carcinoma with Liver Cirrhosis

PubMed Central

Cha, Jung Hoon; Bae, Si Hyun; Kim, Hye Lim; Park, Na Ri; Choi, Eun Suk; Jung, Eun Sun; Choi, Jong Young; Yoon, Seung Kew

2013-01-01

Purpose Recent studies have revealed that branched-chain amino acids (BCAA) reduce the development of hepatocellular carcinoma (HCC) in patients with obesity and hepatitis C virus infection by improving insulin resistance (IR). The aim of this study was to examine the anti-cancer and anti-fibrotic effects of BCAA on the development of diethylnitrosamine (DEN)-induced HCC and liver cirrhosis in a rat model. Methods Male SD rats received weekly intraperitoneal injections of DEN (50 mg/kg of body weight) for 16 weeks to induce HCC. They were fed a diet containing 3% casein, 3% or 6% BCAA for 13 weeks beginning 6 weeks after DEN administration. DEN was used to induce HCC through stepwise development from cirrhosis to HCC. The effect of BCAA was evaluated in tumor tissues by histopathologic analyses, reverse transcription-polymerase chain reaction, and Western blotting. Results The mean area and number of dysplastic nodules (DNs) and tumors in the casein group tended to be larger than those in the BCAA group 16 weeks after DEN administration. The mean fibrotic area in the BCAA group was smaller than that in the casein group. The BCAA group showed decreased mRNA levels for markers of fibrosis, angiogenesis, and apoptosis inhibition. Compared with the casein group, the BCAA group had lower levels of α-smooth muscle actin, vascular endothelial growth factor, p-β-catenin, p-p38 mitogen-activated protein kinase, proliferating cell nuclear antigen, and caspase-3 protein expression, as well as a higher level of cleaved caspase-3 protein expression. Conclusions BCAA supplementation of the diet ameliorated liver fibrosis and HCC development in a DEN-induced rat model of HCC with liver cirrhosis, but not in the IR model. These results provide a rationale for anti-fibrosis and chemoprevention using BCAA treatment for HCC with liver cirrhosis, as well as decreasing the ammonia level. PMID:24223741

76 FR 14407 - Exchange of Letters Between Dr. Murray M. Lumpkin, Deputy Commissioner, International Programs...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-03-16

..., Fisheries and Food of Ireland Concerning Certification Requirements for Caseins, Caseinates, and Mixtures... certification requirements for caseins, caseinates, and mixtures thereof exported from Ireland to the United States. The mutual goals of FDA and DAFF in establishing certification requirements for caseins...

Use of surface plasmon resonance (SPR) to study the dissociation and polysaccharide binding of casein micelles and caseins.

PubMed

Thompson, Abby K; Singh, Harjinder; Dalgleish, Douglas G

2010-11-24

Tests were made to determine whether surface plasmon resonance (SPR) could be used as a technique to study the dissociation properties of bovine casein micelles or of sodium caseinate and the interactions between these protein particles and different polysaccharides. Surfaces of bound micelles or caseinate were made, and the changes in refractive index of these layers were used to define changes in the structures of the chemisorbed material. The technique appears to have some potential for studying details of the dissociation of casein micelles and of the binding of different polysaccharides to caseins.

Chitosan-caseinate bilayer coatings for paper packaging materials.

PubMed

Khwaldia, Khaoula; Basta, Altaf H; Aloui, Hajer; El-Saied, Houssni

2014-01-01

Papers coated with caseinate and caseinate/chitosan bilayer films were developed. Caseinate, chitosan and caseinate/chitosan films were preliminary characterized by FTIR spectroscopy and thermal stability analyses. The effects of coating weight, caseinate concentration (7%, 10%, and 12%, w/w), and coating application methods (single layer and bilayer) on the physical and mechanical properties of coated papers were studied. Increasing the concentration of caseinate led to a decrease in water vapor permeability (WVP) of the resulting coated paper sheets. Chitosan significantly (p<0.05) increased the elongation at break (%E) of coated paper. However, the application of chitosan as a second layer on wet or dry caseinate films did not significantly affect (p>0.05) the tensile strength (TS) of coated paper. The greatest reduction in paper WVP is achieved by addition of a chitosan layer to the dried preformed caseinate-coated paper. Copyright © 2013 Elsevier Ltd. All rights reserved.

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

PubMed

Sakono, Masafumi; Motomura, Konomi; Maruyama, Tatsuo; Kamiya, Noriho; Goto, Masahiro

2011-01-07

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. Copyright © 2010 Elsevier Inc. All rights reserved.

Casein maps: Effect of ethanol, pH, temperature, and CaCl2 on the particle size of reconstituted casein micelles

PubMed Central

Ye, Ran; Harte, Federico

2015-01-01

Although conditions favoring casein micelle aggregation are well known, factors promoting the dissociation of the casein micelle are not fully understood. It was our objective to investigate the ethanol-induced dissociation of micellar casein as affected by temperature and a wide range of pH, along with the concentrations of calcium and casein. Two different concentrations of casein micelles were dispersed in imidazole buffer with 0 to 80% ethanol (vol/vol) and 2 and 10 mM calcium. Apparent micelle size was determined by dynamic light scattering at 5, 30, and 60°C. In the absence of ethanol, casein precipitation occurred at pH 4.6 in imidazole buffer. Ten to forty percent ethanol promoted casein aggregation (>1,000 nm) and higher temperature (30 and 60°C) enhanced this effect. Higher ethanol concentrations at 50 to 80% induced the dissociation (<40 nm) of the casein micelle upon acidification (pH <5) and alkalization (pH >8) in imidazole buffer. In addition, higher concentrations of casein (0.25 mg/mL) and calcium (20 mM) caused the formation of larger aggregates (>1,000 nm) in the presence of ethanol when comparing with the initial lower concentrations of casein (0.1 mg/mL) and calcium (2 mM). Casein micelle dissociation can be achieved near the isoelectric pH by modifying the solvent composition and temperature. PMID:23200467

Casein maps: effect of ethanol, pH, temperature, and CaCl2 on the particle size of reconstituted casein micelles.

PubMed

Ye, Ran; Harte, Federico

2013-02-01

Although conditions favoring casein micelle aggregation are well known, factors promoting the dissociation of the casein micelle are not fully understood. It was our objective to investigate the ethanol-induced dissociation of micellar casein as affected by temperature and a wide range of pH, along with the concentrations of calcium and casein. Two different concentrations of casein micelles were dispersed in imidazole buffer with 0 to 80% ethanol (vol/vol) and 2 and 10mM calcium. Apparent micelle size was determined by dynamic light scattering at 5, 30, and 60°C. In the absence of ethanol, casein precipitation occurred at pH 4.6 in imidazole buffer. Ten to forty percent ethanol promoted casein aggregation (>1,000 nm) and higher temperature (30 and 60°C) enhanced this effect. Higher ethanol concentrations at 50 to 80% induced the dissociation (<40 nm) of the casein micelle upon acidification (pH <5) and alkalization (pH>8) in imidazole buffer. In addition, higher concentrations of casein (0.25mg/mL) and calcium (20mM) caused the formation of larger aggregates (>1,000 nm) in the presence of ethanol when comparing with the initial lower concentrations of casein (0.1mg/mL) and calcium (2mM). Casein micelle dissociation can be achieved near the isoelectric pH by modifying the solvent composition and temperature. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Association of denatured whey proteins with casein micelles in heated reconstituted skim milk and its effect on casein micelle size.

PubMed

Anema, Skelte G; Li, Yuming

2003-02-01

When skim milk at pH 6.55 was heated (75 to 100 degrees C for up to 60 min), the casein micelle size, as monitored by photon correlation spectroscopy, was found to increase during the initial stages of heating and tended to plateau on prolonged heating. At any particular temperature, the casein micelle size increased with longer holding times, and, at any particular holding time, the casein micelle size increased with increasing temperature. The maximum increase in casein micelle size was about 30-35 nm. The changes in casein micelle size were poorly correlated with the level of whey protein denaturation. However, the changes in casein micelle size were highly correlated with the levels of denatured whey proteins that were associated with the casein micelles. The rate of association of the denatured whey proteins with the casein micelles was considerably slower than the rate of denaturation of the whey proteins. Removal of the whey proteins from the skim milk resulted in only small changes in casein micelle size during heating. Re-addition of beta-lactoglobulin to the whey-protein-depleted milk caused the casein micelle size to increase markedly on heat treatment. The changes in casein micelle size induced by the heat treatment of skim milk may be a consequence of the whey proteins associating with the casein micelles. However, these associated whey proteins would need to occlude a large amount of serum to account for the particle size changes. Separate experiments showed that the viscosity changes of heated milk and the estimated volume fraction changes were consistent with the particle size changes observed. Further studies are needed to determine whether the changes in size are due to the specific association of whey proteins with the micelles or whether a low level of aggregation of the casein micelles accompanies this association behaviour. Preliminary studies indicated lower levels of denatured whey proteins associated with the casein micelles and smaller changes in casein micelle size occurred as the pH of the milk was increased from pH 6.5 to pH 6.7.

Milk-induced eczema is associated with the expansion of T cells expressing cutaneous lymphocyte antigen.

PubMed Central

Abernathy-Carver, K J; Sampson, H A; Picker, L J; Leung, D Y

1995-01-01

The extravasation of T cells at sites of inflammation is critically dependent on the activity of homing receptors (HR) involved in endothelial cell recognition and binding. Two such HR (the cutaneous lymphocyte antigen [CLA] and L-selectin) have been shown to be selectively involved in T cell migration to skin and peripheral lymph nodes, respectively. This study was designed to assess the relationship between the organ specificity of an allergic reaction to food and the expression of HR on T cells activated in vitro by the relevant food allergen. Peripheral blood mononuclear cells were isolated from seven milk allergic children with a history of eczema when exposed to milk. All patients had a positive prick skin test and double-blind placebo-controlled food challenge to milk. 10 children with either allergic eosinophilic gastroenteritis or milk-induced enterocolitis and 8 nonatopic adults served as controls. Five-parameter flow cytometry using monoclonal antibodies was used for detection of the specific HR on freshly isolated T cells versus T cell blasts induced by a 6-d incubation with casein, as compared with Candida albicans. After in vitro stimulation with casein, but not C. albicans, patients with milk allergy and atopic dermatitis had a significantly greater percentage of CLA+ T cells (P < 0.01) than controls with milk-induced enterocolitis, allergic eosinophilic gastroenteritis, or nonatopic healthy controls. In contrast, the percentage of L-selectin-expressing T cells did not differ significantly between these groups. These data suggest that after casein stimulation allergic patients with milk-induced skin disease have an expanded population of CLA+ T cells, as compared with nonatopics or allergic patients without skin involvement. We postulate that heterogeneity in the regulation of HR expression on antigen-specific T cells may play a role in determining sites of involvement in tissue-directed allergic responses. Images PMID:7532192

Milk-induced eczema is associated with the expansion of T cells expressing cutaneous lymphocyte antigen.

PubMed

Abernathy-Carver, K J; Sampson, H A; Picker, L J; Leung, D Y

1995-02-01

The extravasation of T cells at sites of inflammation is critically dependent on the activity of homing receptors (HR) involved in endothelial cell recognition and binding. Two such HR (the cutaneous lymphocyte antigen [CLA] and L-selectin) have been shown to be selectively involved in T cell migration to skin and peripheral lymph nodes, respectively. This study was designed to assess the relationship between the organ specificity of an allergic reaction to food and the expression of HR on T cells activated in vitro by the relevant food allergen. Peripheral blood mononuclear cells were isolated from seven milk allergic children with a history of eczema when exposed to milk. All patients had a positive prick skin test and double-blind placebo-controlled food challenge to milk. 10 children with either allergic eosinophilic gastroenteritis or milk-induced enterocolitis and 8 nonatopic adults served as controls. Five-parameter flow cytometry using monoclonal antibodies was used for detection of the specific HR on freshly isolated T cells versus T cell blasts induced by a 6-d incubation with casein, as compared with Candida albicans. After in vitro stimulation with casein, but not C. albicans, patients with milk allergy and atopic dermatitis had a significantly greater percentage of CLA+ T cells (P < 0.01) than controls with milk-induced enterocolitis, allergic eosinophilic gastroenteritis, or nonatopic healthy controls. In contrast, the percentage of L-selectin-expressing T cells did not differ significantly between these groups. These data suggest that after casein stimulation allergic patients with milk-induced skin disease have an expanded population of CLA+ T cells, as compared with nonatopics or allergic patients without skin involvement. We postulate that heterogeneity in the regulation of HR expression on antigen-specific T cells may play a role in determining sites of involvement in tissue-directed allergic responses.

The effects of spaceflight on mammary metabolism in pregnant rats

NASA Technical Reports Server (NTRS)

Plaut, K.; Maple, R.; Vyas, C.; Munaim, S.; Darling, A.; Casey, T.; Alberts, J. R.

1999-01-01

The effects of spaceflight on mammary metabolism of 10 pregnant rats was measured on Day 20 of pregnancy and after parturition. Rats were flown on the space shuttle from Day 11 through Day 20 of pregnancy. After their return to earth, glucose oxidation to carbon dioxide increased 43% (P < 0.05), and incorporation into fatty acids increased 300% (P < 0.005) compared to controls. It is unclear whether the enhanced glucose use is due to spaceflight or a response to landing. Casein mRNA and gross histology were not altered at Day 20 of pregnancy. Six rats gave birth (on Day 22 to 23 of pregnancy) and mammary metabolic activity was measured immediately postpartum. The earlier effects of spaceflight were no longer apparent. There was also no difference in expression of beta-casein mRNA. It is clear from these studies that spaceflight does not impair the normal development of the mammary gland, its ability to use glucose, nor the ability to express mRNA for a major milk protein.

RNA Sequencing of the Human Milk Fat Layer Transcriptome Reveals Distinct Gene Expression Profiles at Three Stages of Lactation

PubMed Central

Lemay, Danielle G.; Ballard, Olivia A.; Hughes, Maria A.; Morrow, Ardythe L.; Horseman, Nelson D.; Nommsen-Rivers, Laurie A.

2013-01-01

Aware of the important benefits of human milk, most U.S. women initiate breastfeeding but difficulties with milk supply lead some to quit earlier than intended. Yet, the contribution of maternal physiology to lactation difficulties remains poorly understood. Human milk fat globules, by enveloping cell contents during their secretion into milk, are a rich source of mammary cell RNA. Here, we pair this non-invasive mRNA source with RNA-sequencing to probe the milk fat layer transcriptome during three stages of lactation: colostral, transitional, and mature milk production. The resulting transcriptomes paint an exquisite portrait of human lactation. The resulting transcriptional profiles cluster not by postpartum day, but by milk Na:K ratio, indicating that women sampled during similar postpartum time frames could be at markedly different stages of gene expression. Each stage of lactation is characterized by a dynamic range (105-fold) in transcript abundances not previously observed with microarray technology. We discovered that transcripts for isoferritins and cathepsins are strikingly abundant during colostrum production, highlighting the potential importance of these proteins for neonatal health. Two transcripts, encoding β-casein (CSN2) and α-lactalbumin (LALBA), make up 45% of the total pool of mRNA in mature lactation. Genes significantly expressed across all stages of lactation are associated with making, modifying, transporting, and packaging milk proteins. Stage-specific transcripts are associated with immune defense during the colostral stage, up-regulation of the machinery needed for milk protein synthesis during the transitional stage, and the production of lipids during mature lactation. We observed strong modulation of key genes involved in lactose synthesis and insulin signaling. In particular, protein tyrosine phosphatase, receptor type, F (PTPRF) may serve as a biomarker linking insulin resistance with insufficient milk supply. This study provides the methodology and reference data set to enable future targeted research on the physiological contributors of sub-optimal lactation in humans. PMID:23861770

Chemical and immunochemical characterization of caseins and the major whey proteins of rabbit milk.

PubMed Central

Dayal, R; Hurlimann, J; Suard, Y M; Kraehenbuhl, J P

1982-01-01

Caseins were separated from whey proteins by acid precipitation of skimmed rabbit milk. Whole casein was resolved by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis into three major bands with apparent relative molecular masses (Mr of 31 000, 29 000 and 25 000. On agarose/urea-gel electrophoresis whole casein gave three bands with electrophoretic mobilities alpha, beta and gamma. The three components were purified by DEAE-cellulose chromatography under denaturing and reducing conditions. Each was shown to have a different amino acid, hexose and phosphorus content, as well as non-identical peptide fragments after proteinase digestion. The 31 000 Da (dalton) protein, of alpha-electrophoretic mobility, had a high phosphorus content (4.38%, w/w); the 29 000 Da peptide, of gamma-mobility, had the highest hexose content (2.2%, w/w), contained 0.8 cysteine residue per 100 amino acid residues and was susceptible to chymosin digestion corresponding thus to kappa-casein; the 25 000 Da protein migrated to the beta-position. The rabbit casein complex is composed of at least three caseins, two of which (alpha- and kappa-caseins) are analogous to the caseins from ruminants. Although caseins are poor immunogens, specific antibodies were raised against total and purified polypeptides. The antiserum directed against whole casein recognized each polypeptide, each casein corresponding to a distinct precipitation line. The antisera directed against each casein polypeptide reacted exclusively with the corresponding casein and no antiserum cross-reaction occurred between the three polypeptides. From whey, several proteins were isolated, characterized and used as antigens to raise specific antibodies. An iron-binding protein with an apparent Mr of 80 000 was shown to be immunologically and structurally identical with serum transferrin. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:6177316

Association of lipids with milk α- and β-caseins.

PubMed

Bourassa, P; Bekale, L; Tajmir-Riahi, H A

2014-09-01

We report the molecular interaction and the binding sites of cholesterol (CHOL), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dioctadecyldimethyl-ammoniumbromide (DDAB), and dioleoylphosphatidylethanolamine (DOPE) with milk α- and β-caseins in aquous solution at physiological conditions. Fourier transform infrared (FTIR), fluorescence spectroscopic methods and molecular modeling were used to determine the binding sites of lipid-protein complexes and the effect of lipid interaction on the stability and conformation of α- and β-caseins. Structural analysis showed that lipids bind casein via mainly hydrophobic contact with association constants of KCHOL-α-casein=1.0 (±0.1)×10(4) M(-1), KDOPE-α-casein=5.0 (±0.07)×10(3) M(-1), KDDAB-α-casein=2.0 (±0.06)×10(4) M(-1), KDOTAP-α-casein=1.5 (±0.6)×10(4) M(-1), KCHOL-β-casein=1.0 (±0.3)×10(4) M(-1), KDOPE-β-casein=1.5 (±0.06)×10(3) M(-1), KDDAB-β-casein=1.7 (±0.3)×10(4) M(-1) and KDOTAP-β-casein=2.1 (±0.5)×10(4) M(-1). The average number of binding sites occupied by lipid molecules on protein (n) were from 0.7 to 1.1. Docking showed different binding sites for α- and β-caseins toward lipid complexation with the free binding energies from -10 to -13 kcal/mol. Casein conformation was altered by lipid interaction with a reduction of α-helix and β-sheet and an increase of random coil and turn structure suggesting a partial protein unfolding. Cascasein; CHOLcholesterol; DOTAP1,2-dioleoyl-3-trimethylammonium-propane; DDABdioctadecyldimethylammonium bromide; DOPEdioleoylphosphatidylethanolamine; FTIRFourier transform infrared spectroscopy; CDcircular dichroism. Copyright © 2014 Elsevier B.V. All rights reserved.

Epigenetic features of FoxP3 in children with cow's milk allergy.

PubMed

Paparo, Lorella; Nocerino, Rita; Cosenza, Linda; Aitoro, Rosita; D'Argenio, Valeria; Del Monaco, Valentina; Di Scala, Carmen; Amoroso, Antonio; Di Costanzo, Margherita; Salvatore, Francesco; Berni Canani, Roberto

2016-01-01

DNA methylation of the Th1 and Th2 cytokine genes is altered during cow's milk allergy (CMA). Forkhead box transcription factor 3 (FoxP3) is essential for the development and function of regulatory T cells (Tregs) and is involved in oral tolerance acquisition. We assessed whether tolerance acquisition in children with IgE-mediated CMA is associated with DNA demethylation of the Treg-specific demethylated region (TSDR) of FoxP3. Forty children (aged 3-18 months) were enrolled: 10 children with active IgE-mediated CMA (group 1), 10 children who outgrew CMA after dietary treatment with an extensively hydrolyzed casein formula containing the probiotic Lactobacillus rhamnosus GG (group 2), 10 children who outgrew CMA after treatment with other formulas (group 3), and 10 healthy controls (group 4). FoxP3 TSDR demethylation and expression were measured in mononuclear cells purified from peripheral blood of the four groups of children. FoxP3 TSDR demethylation was significantly lower in children with active IgE-mediated CMA than in either children who outgrew CMA or in healthy children. Formula selection influenced the FoxP3 TSDR demethylation profile. The FoxP3 TSDR demethylation rate and expression level were correlated. Tolerance acquisition in children with IgE-mediated CMA involves epigenetic regulation of the FoxP3 gene. This feature could be a new target for preventive and therapeutic strategies against CMA.

Phosphorylation Affects DNA-Binding of the Senescence-Regulating bZIP Transcription Factor GBF1

PubMed Central

Smykowski, Anja; Fischer, Stefan M.; Zentgraf, Ulrike

2015-01-01

Massive changes in the transcriptome of Arabidopsis thaliana during onset and progression of leaf senescence imply a central role for transcription factors. While many transcription factors are themselves up- or down-regulated during senescence, the bZIP transcription factor G-box-binding factor 1 (GBF1/bZIP41) is constitutively expressed in Arabidopsis leaf tissue but at the same time triggers the onset of leaf senescence, suggesting posttranscriptional mechanisms for senescence-specific GBF1 activation. Here we show that GBF1 is phosphorylated by the threonine/serine CASEIN KINASE II (CKII) in vitro and that CKII phosphorylation had a negative effect on GBF1 DNA-binding to G-boxes of two direct target genes, CATALASE2 and RBSCS1a. Phosphorylation mimicry at three serine positions in the basic region of GBF1 also had a negative effect on DNA-binding. Kinase assays revealed that CKII phosphorylates at least one serine in the basic domain but has additional phosphorylation sites outside this domain. Two different ckII α subunit1 and one α subunit2 T-DNA insertion lines showed no visible senescence phenotype, but in all lines the expression of the senescence marker gene SAG12 was remarkably diminished. A model is presented suggesting that senescence-specific GBF1 activation might be achieved by lowering the phosphorylation of GBF1 by CKII. PMID:27135347

Serine Protease Zymography: Low-Cost, Rapid, and Highly Sensitive RAMA Casein Zymography.

PubMed

Yasumitsu, Hidetaro

2017-01-01

To detect serine protease activity by zymography, casein and CBB stain have been used as a substrate and a detection procedure, respectively. Casein zymography has been using substrate concentration at 1 mg/mL and employing conventional CBB stain. Although ordinary casein zymography provides reproducible results, it has several disadvantages including time-consuming and relative low sensitivity. Improved casein zymography, RAMA casein zymography, is rapid and highly sensitive. RAMA casein zymography completes the detection process within 1 h after incubation and increases the sensitivity at least by tenfold. In addition to serine protease, the method also detects metalloprotease 7 (MMP7, Matrilysin) with high sensitivity.

Quantitative determination of casein genetic variants in goat milk: Application in Girgentana dairy goat breed.

PubMed

Montalbano, Maria; Segreto, Roberta; Di Gerlando, Rosalia; Mastrangelo, Salvatore; Sardina, Maria Teresa

2016-02-01

The study was conducted to develop a high-performance liquid chromatographic (HPLC) method to quantify casein genetic variants (αs2-, β-, and κ-casein) in milk of homozygous individuals of Girgentana goat breed. For calibration experiments, pure genetic variants were extracted from individual milk samples of animals with known genotypes. The described HPLC approach was precise, accurate and highly suitable for quantification of goat casein genetic variants of homozygous individuals. The amount of each casein per allele was: αs2-casein A = 2.9 ± 0.8 g/L and F = 1.8 ± 0.4 g/L; β-casein C = 3.0 ± 0.8 g/L and C1 = 2.0 ± 0.7 g/L and κ-casein A = 1.6 ± 0.3 g/L and B = 1.1 ± 0.2 g/L. A good correlation was found between the quantities of αs2-casein genetic variants A and F, and β-casein C and C1 with other previously described method. The main important result was obtained for κ-casein because, till now, no data were available on quantification of single genetic variants for this protein. Copyright © 2015 Elsevier Ltd. All rights reserved.

Interaction of lactoferrin and lysozyme with casein micelles.

PubMed

Anema, Skelte G; de Kruif, C G Kees

2011-11-14

On addition of lactoferrin (LF) to skim milk, the turbidity decreases. The basic protein binds to the caseins in the casein micelles, which is then followed by a (partial) disintegration of the casein micelles. The amount of LF initially binding to casein micelles follows a Langmuir adsorption isotherm. The kinetics of the binding of LF could be described by first-order kinetics and similarly the disintegration kinetics. The disintegration was, however, about 10 times slower than the initial adsorption, which allowed investigating both phenomena. Kinetic data were also obtained from turbidity measurements, and all data could be described with one equation. The disintegration of the casein micelles was further characterized by an activation energy of 52 kJ/mol. The initial increase in hydrodynamic size of the casein micelles could be accounted for by assuming that it would go as the cube root of the mass using the adsorption and disintegration kinetics as determined from gel electrophoresis. The results show that LF binds to casein micelles and that subsequently the casein micelles partly disintegrate. All micelles behave in a similar manner as average particle size decreases. Lysozyme also bound to the casein micelles, and this binding followed a Langmuir adsorption isotherm. However, lysozyme did not cause the disintegration of the casein micelles.

Lipid oxidation in algae oil-in-water emulsions stabilized by bovine and caprine caseins

USDA-ARS?s Scientific Manuscript database

Caseins (alpha S1-, alpha S2-, and beta-casein) are phosphoproteins that are capable of binding transition metals and scavenging free radicals, these properties make them good candidates to be used as natural antioxidants in oil-in-water emulsions. Caprine casein exhibits variability in aS1-casein c...

Casein kinase II induces c-fos expression via the serum response element pathway and p67SRF phosphorylation in living fibroblasts.

PubMed Central

Gauthier-Rouvière, C; Basset, M; Blanchard, J M; Cavadore, J C; Fernandez, A; Lamb, N J

1991-01-01

Elevation of intracellular casein kinase II (CKII) levels through microinjection of purified CKII results in the rapid and transient induction of c-fos in quiescent rat embryo fibroblasts, and activation of quiescent cells by serum is accompanied by the nuclear relocation of endogenous CKII. The induction of c-fos by CKII is inhibited by coinjection of oligonucleotides corresponding to the sequence of the serum response element (SRE) present in the c-fos promoter, indicating that competitive displacement of positive factors from the endogenous c-fos SRE prevents c-fos induction by CKII. Furthermore, the expression of c-fos induced by either CKII injection or serum activation is also inhibited by microinjection of antibodies against the 67 kDa serum response factor (p67SRF) indicating the absolute requirement of p67SRF in this process. Finally, we show the specific phosphorylation of p67SRF in vivo following microinjection of CKII into quiescent cells. Together, these data strongly support that CKII induces c-fos expression through binding/activation of the phosphorylated p67SRF at the SRE sequence. Images PMID:1915270

Estrogenic status modulates the effect of soy on hepatic responses to 7,12-dimethylbenz(a)anthracene (DMBA)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Singhal, Rohit; Badger, Thomas M.; Arkansas Children's Nutrition Center, Little Rock, AR-72202

2009-01-01

We examined the influence of estradiol (E2) status and soy protein isolate (SPI) intake on the hepatic responses altered by 7,12-dimethylbenz(a)anthracene (DMBA, a polycyclic aromatic hydrocarbon [PAH]). Sprague-Dawley rats were ovariectomized (OVX) at PND50 and infused with E2 or vehicle for 14 d and gavaged with 50 mg/kg DMBA or vehicle 24 h before sacrifice at PND64. Rats were fed an AIN-93G diet made with SPI or casein as sole protein source throughout the study. Basal AhR protein levels were reduced (P < 0.05) by SPI feeding irrespective of the E2 status. However, DMBA increased (P < 0.05) AhR-induced CYP1A1more » gene expression in OVX, SPI-fed rats, but reduced (P < 0.05) CYP1A1 in OVX + E2, SPI-fed rats. Chromatin-immunoprecipitation demonstrated lower (P < 0.05) DMBA-mediated recruitment of estrogen receptor alpha to the CYP1A1 promoter by SPI feeding in the presence of E2, suggesting an estrogen-like action of SPI on DMBA-mediated signaling in the absence of E2. Further, microarray analysis (Rat 230-2.0 Affymetrix-GeneChip{sup TM}) revealed 231 genes common to SPI + DMBA and SPI + E2 + DMBA (normalized to E2) treatments. AhR-activated genes (CYP1A1, CYP1A2, and NQO1) were down-regulated by SPI + E2 + DMBA compared to SPI + DMBA. Unique interactions among SPI, DMBA and E2 altered the expression profile of 316 genes, not observed by either treatment alone. Our data suggest that although E2 status does not effect soy-mediated AhR degradation, it modulates the effects of soy on many genes, including CYP1A1.« less

A functional study of proximal goat β-casein promoter and intron 1 in immortalized goat mammary epithelial cells.

PubMed

Kung, M H; Lee, Y J; Hsu, J T; Huang, M C; Ju, Y T

2015-06-01

Goat β-casein (CSN2) promoter has been extensively used to derive expression of recombinant therapeutic protein in transgenic goats; however, little direct evidence exists for signaling molecules and the cis-elements of goat CSN2 promoter in response to lactogenic hormone stimulation in goat mammary epithelial cells. Here, we use an immortalized caprine mammary epithelial cell line (CMC) to search for evidence of the above. Serial 5'-flanking regions deleted of promoter and intron 1 in goat CSN2 (-4,047 to +2,054) driven by firefly luciferase reporter gene were constructed and applied to measure promoter activity in CMC. The intron 1 region (+393 to +501) significantly decreased basal activity of the promoter. This finding contradicts other studies of the role of intron 1. The signal transducer and activator of transcription (STAT)5a played a significant role in activating promoter activity by prolactin stimulation. Hydrocortisone enhanced and prolonged the activity of STAT5a and promoter in CMC, but was independent of the glucocorticoid receptor response element. The minimum length of the CSN2 promoter segment in response to lactogenic stimulation was confirmed by 5' serial deletions. A cis-element located from -300 to -90 in proximal goat CSN2 promoter that is absent in bovine and human CSN2 promoter was newly identified. We demonstrated the presence of a STAT5a binding site (-102 to -82) and preservation of the guanosine nucleotide at position -90 based on responses to the presence of lactogenic hormone using internal deletions and point mutations of the predicted STAT5a binding site, and chromatin immunoprecipitation assay. Together, these findings demonstrate that the proximal -300 bp of goat CSN2 promoter containing the STAT5a binding site (-102 to -82) is the response element for lactogenic hormone stimulation. Additionally, intron 1 may be required for tissue or developmental stage-specific expression in mammary gland. The role of the far-distal regions of goat CSN2 promoter in high-level lactogenic hormone induction and specific expression require further examination. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Structure, chaperone-like activity and allergenicity profile of bovine caseins upon peroxynitrite modification: New evidences underlying mastitis pathomechanisms.

PubMed

Sadeghian, Tanaz; Tavaf, Zohreh; Oryan, Ahmad; Shokouhi, Raheleh; Pourpak, Zahra; Moosavi-Movahedi, Ali Akbar; Yousefi, Reza

2018-01-01

Mastitis, an inflammatory reaction frequently develops in response to intra-mammary bacterial infection‌‌, may induce the generation of peroxynitrite (PON)‌ which is a highly potent reactive oxygen and nitrogen species. Caseins as the intrinsically unfolded proteins seem feasible substrates to react with PON. Therefore, in the current study, structural and functional aspects of both β-casein (β-CN) and whole casein fraction (WCF) were evaluated after PON modification, using a variety of techniques. Modification of the bovine caseins with PON results in an important enhancement in the carbonyl, nitrotryptophan, nitrotyrosine and dityrosine content of these proteins‌. The results of fluorescence and far UV-CD assessments suggested significant structural alteration of caseins upon PON-modification. The chaperone-like activity of β-casein was significantly altered after PON modification. The results of scanning electron microscopy suggest that bovine caseins display unique morphological features after treatment with PON. Also, the PON-modified caseins preserved their allergenicity profile and displayed partial resistance against digestion by the pancreatic proteases. Ascorbic acid, an important antioxidant component of milk, was also capable to significantly prevent the PON-induced structural damages in bovine milk caseins. In conclusion, our results suggest that PON may have significant role in the structural and functional alteration of milk caseins. Also, the PON-induced structural damaging effects of caseins might be effectively prevented by a sufficient level of milk antioxidant components particularly by ascorbic acid. Copyright © 2017 Elsevier B.V. All rights reserved.

Apical-to-basolateral transepithelial transport of cow's milk caseins by intestinal Caco-2 cell monolayers: MS-based quantitation of cellularly degraded α- and β-casein fragments.

PubMed

Sakurai, Nao; Nishio, Shunsuke; Akiyama, Yuka; Miyata, Shinji; Oshima, Kenzi; Nadano, Daita; Matsuda, Tsukasa

2018-02-27

Casein is the major milk protein to nourish infants but, in certain population, it causes cow's milk allergy, indicating the uptake of antigenic casein and their peptides through the intestinal epithelium. Using human intestinal Caco-2 cell monolayers, the apical-to-basal transepithelial transport of casein was investigated. Confocal microscopy using component-specific antibodies showed that αs1-casein antigens became detectable as punctate signals at the apical-side cytoplasm and reached to the cytoplasm at a tight-junction level within a few hours. Such intracellular casein signals were more remarkable than those of the other antigens, β-lactoglobulin and ovalbumin, colocalized in part with an early endosome marker protein, EEA1, and decreased in the presence of cytochalasin D or sodium azide and also at lowered temperature at 4 °C. LC-MS analysis of the protein fraction in the basal-side medium identified the αs1-casein fragment including the N-terminal region and the αs2-casein fragment containing the central part of polypeptide at 100∼1000 fmol per well levels. Moreover, β-casein C-terminal overlapping peptides were identified in the peptide fraction below 10 kDa of the basal medium. These results suggest that caseins are partially degraded by cellular proteases and/or peptidases and immunologically active casein fragments are transported to basal side of the cell monolayers.

Nutritional Regulation of Bile Acid Metabolism Is Associated with Improved Pathological Characteristics of the Metabolic Syndrome*

PubMed Central

Liaset, Bjørn; Hao, Qin; Jørgensen, Henry; Hallenborg, Philip; Du, Zhen-Yu; Ma, Tao; Marschall, Hanns-Ulrich; Kruhøffer, Mogens; Li, Ruiqiang; Li, Qibin; Yde, Christian Clement; Criales, Gabriel; Bertram, Hanne C.; Mellgren, Gunnar; Øfjord, Erik Snorre; Lock, Erik-Jan; Espe, Marit; Frøyland, Livar; Madsen, Lise; Kristiansen, Karsten

2011-01-01

Bile acids (BAs) are powerful regulators of metabolism, and mice treated orally with cholic acid are protected from diet-induced obesity, hepatic lipid accumulation, and increased plasma triacylglycerol (TAG) and glucose levels. Here, we show that plasma BA concentration in rats was elevated by exchanging the dietary protein source from casein to salmon protein hydrolysate (SPH). Importantly, the SPH-treated rats were resistant to diet-induced obesity. SPH-treated rats had reduced fed state plasma glucose and TAG levels and lower TAG in liver. The elevated plasma BA concentration was associated with induction of genes involved in energy metabolism and uncoupling, Dio2, Pgc-1α, and Ucp1, in interscapular brown adipose tissue. Interestingly, the same transcriptional pattern was found in white adipose tissue depots of both abdominal and subcutaneous origin. Accordingly, rats fed SPH-based diet exhibited increased whole body energy expenditure and heat dissipation. In skeletal muscle, expressions of the peroxisome proliferator-activated receptor β/δ target genes (Cpt-1b, Angptl4, Adrp, and Ucp3) were induced. Pharmacological removal of BAs by inclusion of 0.5 weight % cholestyramine to the high fat SPH diet attenuated the reduction in abdominal obesity, the reduction in liver TAG, and the decrease in nonfasted plasma TAG and glucose levels. Induction of Ucp3 gene expression in muscle by SPH treatment was completely abolished by cholestyramine inclusion. Taken together, our data provide evidence that bile acid metabolism can be modulated by diet and that such modulation may prevent/ameliorate the characteristic features of the metabolic syndrome. PMID:21680746

Interactions of milk α- and β-casein with malvidin-3-O-glucoside and their effects on the stability of grape skin anthocyanin extracts.

PubMed

He, Zhiyong; Xu, Mingzhu; Zeng, Maomao; Qin, Fang; Chen, Jie

2016-05-15

The interactions of α- and β-casein with malvidin-3-O-glucoside (MG), the major anthocyanin in grape skin anthocyanin extracts (GSAE), were examined at pH 6.3 by fluorescence, fourier transform infrared (FTIR) and circular dichroism (CD) spectroscopy. The binding constant (KS), binding force and effects of the interactions on the caseins conformation and GSAE stability were investigated. The results showed that α- and β-casein bound with MG via hydrophilic (van der Waals forces or hydrogen bonding) and hydrophobic interactions, respectively. α-Casein had a slightly stronger binding affinity toward MG than β-casein, with respective KS values of 0.51×10(3)M(-1) and 0.46×10(3)M(-1) at 297K. The secondary structures of α- and β-casein were changed by MG binding, with a decrease in α-helix and an increase in turn for α-casein and no change in α-helix and a decrease in turn for β-casein. The casein-anthocyanin interaction appeared to have a positive effect on the thermal, oxidation and photo stability of GSAE. Copyright © 2015 Elsevier Ltd. All rights reserved.

Glycation Reactions of Casein Micelles.

PubMed

Moeckel, Ulrike; Duerasch, Anja; Weiz, Alexander; Ruck, Michael; Henle, Thomas

2016-04-13

After suspensions of micellar casein or nonmicellar sodium caseinate had been heated, respectively, in the presence and absence of glucose for 0-4 h at 100 °C, glycation compounds were quantitated. The formation of Amadori products as indicators for the "early" Maillard reaction were in the same range for both micellar and nonmicellar caseins, indicating that reactive amino acid side chains within the micelles are accessible for glucose in a comparable way as in nonmicellar casein. Significant differences, however, were observed concerning the formation of the advanced glycation end products (AGEs), namely, N(ε)-carboxymethyllysine (CML), pyrraline, pentosidine, and glyoxal-lysine dimer (GOLD). CML could be observerd in higher amounts in nonmicellar casein, whereas in the micelles the pyrraline formation was increased. Pentosidine and GOLD were formed in comparable amounts. Furthermore, the extent of protein cross-linking was significantly higher in the glycated casein micelles than in the nonmicellar casein samples. Dynamic light scattering and scanning electron microscopy showed that glycation has no influence on the size of the casein micelles, indicating that cross-linking occurs only in the interior of the micelles, but altered the surface morphology. Studies on glycation and nonenzymatic cross-linking can contribute to the understanding of the structure of casein micelles.

Prior lactose glycation of caseinate via the Maillard reaction affects in vitro activities of the pepsin-trypsin digest toward intestinal epithelial cells.

PubMed

Wang, X P; Zhao, X H

2017-07-01

The well-known Maillard reaction in milk occurs between lactose and milk proteins during thermal treatment, and its effects on milk nutrition and safety have been well studied. A lactose-glycated caseinate was prepared via this reaction and digested using 2 digestive proteases, pepsin and trypsin. The glycated caseinate digest was assessed for its in vitro activities on rat intestinal epithelial cells in terms of growth proliferation, anti-apoptotic effect, and differentiation induction using caseinate digest as reference, to verify potential effects of the Maillard reaction on these activities of caseinate digest to the cells. Two digests had proliferative and anti-apoptotic effects, and reached the highest effects at 0.02 g/L of digest concentration with treatment time of 24 h. In comparison with caseinate digest, glycated caseinate digest always showed weaker proliferative (5.3-14.2%) and anti-apoptotic (5.9-39.0%) effects, and was more toxic to the cells at 0.5 g/L of digest concentration with treatment time of 48 h. However, glycated caseinate digest at 2 incubation times of 4 to 7 d showed differentiation induction higher than caseinate digest, as it could confer the cells with increased activities in lactase (16.3-26.6%), sucrase (22.4-31.2%), and alkaline phosphatase (17.4-24.8%). Transmission electron microscopy observation results also confirmed higher differentiation induction of glycated caseinate digest. Amino acid loss and lactose glycation partially contributed to these decreased and enhanced activities of glycated caseinate digest, respectively. The Maillard reaction of caseinate and lactose is thus shown in this study to have effects on the activities of caseinate digest to intestinal epithelial cells. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Expression of aquaporins in the efferent ductules, sperm counts, and sperm motility in estrogen receptor-alpha deficient mice fed lab chow versus casein.

PubMed

Ruz, Ricardo; Gregory, Mary; Smith, Charles E; Cyr, Daniel G; Lubahn, Dennis B; Hess, Rex A; Hermo, Louis

2006-02-01

Estrogens play an important role in the male reproductive tract, and this is especially so for the efferent ductules, where alpha-estrogen receptors (ERalpha) have been localized. Mice deficient in ERalpha (alphaERKO mice) are infertile, and the effect appears to be due in part to retention of water at the level of the efferent ductules. In the present study, we examined the consequences of ERalpha deletion on the distribution of certain aquaporins (AQPs), water protein channels, in the efferent ductules and on sperm numbers and motility. In addition, the effects of feeding mice a regular lab chow diet, which contains phytoestrogens, known to affect male reproductive tract functions, and a casein diet, which lacks phytoestrogens, were also assessed. Light microscope immunolocalizations of AQP-1 and AQP-9 revealed dramatic reduction and patchier staining in alphaERKO mice with distal areas of the efferent ductules being more affected than proximal areas. No other changes in immunolocalizations were noted as a consequence of diet. Computer-assisted sperm analyses demonstrated a 62% reduction in cauda epididymal sperm/ml in alphaERKO mice fed lab chow, whereas 87% fewer sperm/ml were observed in alphaERKO mice fed casein, suggesting an enhanced role for sperm production and concentration in a diet containing phytoestrogens. All sperm motility parameters were altered to some degree in alphaERKO mice fed lab chow. Alterations in sperm motility parameters were also detected, but were less dramatic in alphaERKO mice fed casein. These data suggest that the decrease in AQP expression in the efferent ductules of alphaERKO mice contributes in part to water retention in this tissue, eventually leading to backflow of water into the testis, with subsequent decreases in sperm concentration and motility. The data also suggest that phytoestrogens, which are present in regular lab chow, can influence the male reproductive tract with and without the presence of ERalpha, promoting efferent ductule and epididymal functions when ERalpha is expressed, but inhibiting these same functions when ERalpha is missing. Taken together the data underscore the importance of estrogens and ERalpha in maintaining sperm maturation and preventing male infertility. (c) 2005 Wiley-Liss, Inc.

Invited review: Caseins and the casein micelle: their biological functions, structures, and behavior in foods.

PubMed

Holt, C; Carver, J A; Ecroyd, H; Thorn, D C

2013-10-01

A typical casein micelle contains thousands of casein molecules, most of which form thermodynamically stable complexes with nanoclusters of amorphous calcium phosphate. Like many other unfolded proteins, caseins have an actual or potential tendency to assemble into toxic amyloid fibrils, particularly at the high concentrations found in milk. Fibrils do not form in milk because an alternative aggregation pathway is followed that results in formation of the casein micelle. As a result of forming micelles, nutritious milk can be secreted and stored without causing either pathological calcification or amyloidosis of the mother's mammary tissue. The ability to sequester nanoclusters of amorphous calcium phosphate in a stable complex is not unique to caseins. It has been demonstrated using a number of noncasein secreted phosphoproteins and may be of general physiological importance in preventing calcification of other biofluids and soft tissues. Thus, competent noncasein phosphoproteins have similar patterns of phosphorylation and the same type of flexible, unfolded conformation as caseins. The ability to suppress amyloid fibril formation by forming an alternative amorphous aggregate is also not unique to caseins and underlies the action of molecular chaperones such as the small heat-shock proteins. The open structure of the protein matrix of casein micelles is fragile and easily perturbed by changes in its environment. Perturbations can cause the polypeptide chains to segregate into regions of greater and lesser density. As a result, the reliable determination of the native structure of casein micelles continues to be extremely challenging. The biological functions of caseins, such as their chaperone activity, are determined by their composition and flexible conformation and by how the casein polypeptide chains interact with each other. These same properties determine how caseins behave in the manufacture of many dairy products and how they can be used as functional ingredients in other foods. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Milk caseins as useful vehicle for delivery of dipyridamole drug.

PubMed

Dezhampanah, Hamid; Esmaili, Masoomeh; Hasani, Leila

2018-05-01

The interaction of bovine milk α- and β-caseins as an efficient drug carrier system with Dipyridamole (DIP) was investigated using spectroscopy and molecular docking studies at different temperatures (20-37 °C). FTIR, CD, and fluorescence spectroscopy methods demonstrated that α- and β-caseins interact with DIP molecule mainly via hydrophobic and hydrophilic interactions and change in secondary structure of α- and β-caseins. DIP showed a higher quenching efficiency and binding constant of α-casein than β-casein. There was only one binding site for DIP and it was located on the surface of the protein molecule. The thermodynamic parameters of calculation showed that the binding process occurs spontaneously and demonstrated that α- and β-caseins provide very good binding and entrapment to DIP via hydrogen bonds, Van der Waals forces, and hydrophobic interactions. Fluorescence resonance energy transfer, synchronous fluorescence spectroscopy, and docking study showed that DIP binds to the Trp residues of α- and β-casein molecules with short distances. Docking study showed that DIP molecule made several hydrogen bonds and van der Waals interactions with α- and β-caseins. The study of cell culture and micellar solubility of DIP demonstrated α- and β-caseins relatively the same helping in delivery of DIP. Milk α- and β-caseins are considered as a useful vehicle for the solublization and stabilization of DIP in aqueous solution at natural pH.

Effects of Surface Passivation on Gliding Motility Assays

PubMed Central

Maloney, Andy; Herskowitz, Lawrence J.; Koch, Steven J.

2011-01-01

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. PMID:21674032

Casein SNP in Norwegian goats: additive and dominance effects on milk composition and quality

PubMed Central

2011-01-01

Background The four casein proteins in goat milk are encoded by four closely linked casein loci (CSN1S1, CSN2, CSN1S2 and CSN3) within 250 kb on caprine chromosome 6. A deletion in exon 12 of CSN1S1, so far reported only in Norwegian goats, has been found at high frequency (0.73). Such a high frequency is difficult to explain because the national breeding goal selects against the variant's effect. Methods In this study, 575 goats were genotyped for 38 Single Nucleotide Polymorphisms (SNP) located within the four casein genes. Milk production records of these goats were obtained from the Norwegian Dairy Goat Control. Test-day mixed models with additive and dominance fixed effects of single SNP were fitted in a model including polygenic effects. Results Significant additive effects of single SNP within CSN1S1 and CSN3 were found for fat % and protein %, milk yield and milk taste. The allele with the deletion showed additive and dominance effects on protein % and fat %, and overdominance effects on milk quantity (kg) and lactose %. At its current frequency, the observed dominance (overdominance) effects of the deletion allele reduced its substitution effect (and additive genetic variance available for selection) in the population substantially. Conclusions The selection pressure of conventional breeding on the allele with the deletion is limited due to the observed dominance (overdominance) effects. Inclusion of molecular information in the national breeding scheme will reduce the frequency of this deletion in the population. PMID:21864407

Valsartan Upregulates Kir2.1 in Rats Suffering from Myocardial Infarction via Casein Kinase 2.

PubMed

Li, Xinran; Hu, Hesheng; Wang, Ye; Xue, Mei; Li, Xiaolu; Cheng, Wenjuan; Xuan, Yongli; Yin, Jie; Yang, Na; Yan, Suhua

2015-06-01

Myocardial infarction (MI) results in an increased susceptibility to ventricular arrhythmias, due in part to decreased inward-rectifier K+ current (IK1), which is mediated primarily by the Kir2.1 protein. The use of renin-angiotensin-aldosterone system antagonists is associated with a reduced incidence of ventricular arrhythmias. Casein kinase 2 (CK2) binds and phosphorylates SP1, a transcription factor of KCNJ2 that encodes Kir2.1. Whether valsartan represses CK2 activation to ameliorate IK1 remodeling following MI remains unclear. Wistar rats suffering from MI received either valsartan or saline for 7 days. The protein levels of CK2 and Kir2.1 were each detected via a Western blot analysis. The mRNA levels of CK2 and Kir2.1 were each examined via quantitative real-time PCR. CK2 expression was higher at the infarct border; and was accompanied by a depressed IK1/Kir2.1 protein level. Additionally, CK2 overexpression suppressed KCNJ2/Kir2.1 expression. By contrast, CK2 inhibition enhanced KCNJ2/Kir2.1 expression, establishing that CK2 regulates KCNJ2 expression. Among the rats suffering from MI, valsartan reduced CK2 expression and increased Kir2.1 expression compared with the rats that received saline treatment. In vitro, hypoxia increased CK2 expression and valsartan inhibited CK2 expression. The over-expression of CK2 in cells treated with valsartan abrogated its beneficial effect on KCNJ2/Kir2.1. AT1 receptor antagonist valsartan reduces CK2 activation, increases Kir2.1 expression and thereby ameliorates IK1 remodeling after MI in the rat model.

Impact of Nutritional Factors on the Proteome of Intestinal Escherichia coli: Induction of OxyR-Dependent Proteins AhpF and Dps by a Lactose-Rich Diet

PubMed Central

Rothe, Monique; Alpert, Carl; Engst, Wolfram; Musiol, Stephanie; Loh, Gunnar

2012-01-01

To study the impact of nutritional factors on protein expression of intestinal bacteria, gnotobiotic mice monoassociated with Escherichia coli K-12 were fed three different diets: a diet rich in starch, a diet rich in nondigestible lactose, and a diet rich in casein. Two-dimensional gel electrophoresis and electrospray-tandem mass spectrometry were used to identify differentially expressed proteins of bacteria recovered from small intestine and cecum. Oxidative stress response proteins such as AhpF, Dps, and Fur, all of which belong to the oxyR regulon, were upregulated in E. coli isolates from mice fed the lactose-rich diet. Luciferase reporter gene assays demonstrated that osmotic stress caused by carbohydrates led to the expression of ahpCF and dps, which was not observed in an E. coli ΔoxyR mutant. Growth of ahpCF and oxyR deletion mutants was strongly impaired when nondigestible sucrose was present in the medium. The wild-type phenotype could be restored by complementation of the deletions with plasmids containing the corresponding genes and promoters. The results indicate that some OxyR-dependent proteins play a major role in the adaptation of E. coli to osmotic stress. We conclude that there is an overlap of osmotic and oxidative stress responses. Mice fed the lactose-rich diet possibly had a higher intestinal osmolality, leading to the upregulation of OxyR-dependent proteins, which enable intestinal E. coli to better cope with diet-induced osmotic stress. PMID:22427493

Hypoallergenic and Physicochemical Properties of the A2 β-Casein Fractionof Goat Milk.

PubMed

Jung, Tae-Hwan; Hwang, Hyo-Jeong; Yun, Sung-Seob; Lee, Won-Jae; Kim, Jin-Wook; Ahn, Ji-Yun; Jeon, Woo-Min; Han, Kyoung-Sik

2017-01-01

Goat milk has a protein composition similar to that of breast milk and contains abundant nutrients, but its use in functional foods is rather limited in comparison to milk from other sources. The aim of this study was to prepare a goat A2 β-casein fraction with improved digestibility and hypoallergenic properties. We investigated the optimal conditions for the separation of A2 β-casein fraction from goat milk by pH adjustment to pH 4.4 and treating the casein suspension with calcium chloride (0.05 M for 1 h at 25°C). Selective reduction of β-lactoglobulin and α s -casein was confirmed using sodium dodecyl sulphate-polyacrylamide gel electrophoresis and reverse-phase high-performance liquid chromatography. The hypoallergenic property of A2 β-casein fraction was examined by measuring the release of histamine and tumor necrosis factor alpha from HMC-1 human mast cells exposed to different proteins, including A2 β-casein fraction. There was no significant difference in levels of both indicators between A2 β-casein treatment and the control (no protein treatment). The A2 β-casein fraction is abundant in essential amino acids, especially, branched-chain amino acids (leucine, valine, and isoleucine). The physicochemical properties of A2 β-casein fraction, including protein solubility and viscosity, are similar to those of bovine whole casein which is widely used as a protein source in various foods. Therefore, the goat A2 β-casein fraction may be useful as a food material with good digestibility and hypoallergenic properties for infants, the elderly, and people with metabolic disorders.

Some rheological properties of sodium caseinate-starch gels.

PubMed

Bertolini, Andrea C; Creamer, Lawrence K; Eppink, Mieke; Boland, Mike

2005-03-23

The influence of sodium caseinate on the thermal and rheological properties of starch gels at different concentrations and from different botanical sources was evaluated. In sodium caseinate-starch gels, for all starches with the exception of potato starch, the sodium caseinate promoted an increase in the storage modulus and in the viscosity of the composite gel when compared with starch gels. The addition of sodium caseinate resulted in an increase in the onset temperature, the gelatinization temperature, and the end temperature, and there was a significant interaction between starch and sodium caseinate for the onset temperature, the peak temperature, and the end temperature. Microscopy results suggested that sodium caseinate promoted an increase in the homogeneity in the matrix of cereal starch gels.

Interaction between casein micelles and whey protein/κ-casein complexes during renneting of heat-treated reconstituted skim milk powder and casein micelle/serum mixtures.

PubMed

Kethireddipalli, Prashanti; Hill, Arthur R; Dalgleish, Douglas G

2011-02-23

Casein micelles were separated from unheated reconstituted skim milk powder (RSMP) and were resuspended in the serum of RSMP that had been heated, with and without dialysis of this serum against unheated RSMP. Using size-exclusion chromatography, it was found that the soluble complexes of whey protein (WP) with κ-casein in the serum of the heated milk bind progressively to unheated casein micelles during renneting, even prior to the onset of clotting. Similar trends were noted when casein micelles from RSMP heated at pH values of 6.7, 7.1, or 6.3, each with different amounts of WP coating the micelles, were renneted in the presence of soluble WP/κ-casein complexes. No matter what was the initial load of micelle-bound WP complexes, all micelle types were capable of binding additional serum protein complexes during renneting. However, it is not clear that this binding of WP/κ-casein complexes to the micellar surface is a direct cause of the impaired rennet clotting of the RSMP.

Enhanced bioethanol production from pretreated corn stover via multi-positive effect of casein micelles.

PubMed

Eckard, Anahita Dehkhoda; Muthukumarappan, Kasiviswanathan; Gibbons, William

2013-05-01

Casein polypeptides containing substructures of αs1-casein, β-casein, k-casein, αs2-casein were used as a lignin-blocker at above critical micelles concentration to improve the bioethanol production of dilute acid, lime, alkali, extrusion and AFEX pretreated corn stover (CS). Application of 0.5 g/g glucan of casein was found to effectively increase the glucose yield of CS pretreated with dilute acid, lime, alkali, extrusion and AFEX by 31.9%, 17.0%, 22.7%, 29.5%, and 17.4%, respectively with no positive impact on Avicel. Consequently 96 h simultaneous saccharification and fermentation (SSF) of these hydrolysates reduced the fermentation period by up to 48 h and increased the theoretical yield of ethanol by 8.48-33.7% compared to control. Application of casein during saccharification reduced the enzyme utilization by 33.0%. Recycling of hydrolysate from casein-treated CS for a 2nd round hydrolysis resulted in average glucose yield of 36.4% compared to 29.0% control. Copyright © 2012 Elsevier Ltd. All rights reserved.

Investigating cold gelation properties of recombined highly concentrated micellar casein concentrate and cream for use in cheese making.

PubMed

Lu, Y; McMahon, D J; Vollmer, A H

2016-07-01

Highly concentrated micellar casein concentrate (HC-MCC), a potential ingredient for cheese making, contains ~20% casein with ~70% of serum proteins removed by microfiltration and diafiltration of skim milk, followed by vacuum evaporation. Our objective was to investigate cold gelation properties of recombined concentrated milk (RCM) by mixing thawed frozen HC-MCC and cream under different casein levels, pH, and protein-to-fat ratios, and with addition of sodium citrate or calcium. The HC-MCC was recombined with cream using low shear at 50°C for 30 min, and rheological measurements were conducted. Cold-gelling temperature [the temperature at which storage modulus (G')=loss modulus (G″)] was linearly correlated with casein levels from 8.6 to 11.5% (R(2)=0.71), pH from 6.6 to 7.0 (R(2)=0.96), and addition of sodium citrate from 0 to 0.36mmol/g of casein (R(2)=0.80). At pH 7.0, gelation occurred at 12, 26, and 38°C with 9, 10, and 11% casein, respectively. At pH 6.6, 6.8, and 7.0, RCM with 12% casein gelled at a mean temperature of 12, 26, and 37°C, respectively. Adding calcium chloride at 0.17mmol/g of casein significantly increased cold-gelling temperature from 18 to ≥50°C, whereas no significant change was observed at levels up to 0.12mmol/g of casein. Different protein to fat ratios ranging from 0.8 to 1.2 did not significantly influence gelling temperature. In transmission electron micrographs of RCM with 12% casein, casein micelles were nonspherical and partially dissociated into small protein strands. Upon addition of calcium chloride at 0.21mmol/g of casein, casein micelles were more spherical and retained colloidal structure with the presence of aggregated casein micelles. These gelation processes of RCM with or without addition of trisodium citrate were both reversible. We propose that cold gelation of RCM occurs when protein strands that have been partially released from the casein micelles entangle, restrict their mobility, and form a fine-stranded gel network. Upon addition of high levels of calcium, cold gelation was promoted presumably through direct aggregation of casein micelles. Understanding cold gelation properties can facilitate potential use of RCM in cheese making. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

A colostrum trypsin inhibitor gene expressed in the Cape fur seal mammary gland during lactation.

PubMed

Pharo, Elizabeth A; Cane, Kylie N; McCoey, Julia; Buckle, Ashley M; Oosthuizen, W H; Guinet, Christophe; Arnould, John P Y

2016-03-01

The colostrum trypsin inhibitor (CTI) gene and transcript were cloned from the Cape fur seal mammary gland and CTI identified by in silico analysis of the Pacific walrus and polar bear genomes (Order Carnivora), and in marine and terrestrial mammals of the Orders Cetartiodactyla (yak, whales, camel) and Perissodactyla (white rhinoceros). Unexpectedly, Weddell seal CTI was predicted to be a pseudogene. Cape fur seal CTI was expressed in the mammary gland of a pregnant multiparous seal, but not in a seal in its first pregnancy. While bovine CTI is expressed for 24-48 h postpartum (pp) and secreted in colostrum only, Cape fur seal CTI was detected for at least 2-3 months pp while the mother was suckling its young on-shore. Furthermore, CTI was expressed in the mammary gland of only one of the lactating seals that was foraging at-sea. The expression of β-casein (CSN2) and β-lactoglobulin II (LGB2), but not CTI in the second lactating seal foraging at-sea suggested that CTI may be intermittently expressed during lactation. Cape fur seal and walrus CTI encode putative small, secreted, N-glycosylated proteins with a single Kunitz/bovine pancreatic trypsin inhibitor (BPTI) domain indicative of serine protease inhibition. Mature Cape fur seal CTI shares 92% sequence identity with Pacific walrus CTI, but only 35% identity with BPTI. Structural homology modelling of Cape fur seal CTI and Pacific walrus trypsin based on the model of the second Kunitz domain of human tissue factor pathway inhibitor (TFPI) and porcine trypsin (Protein Data Bank: 1TFX) confirmed that CTI inhibits trypsin in a canonical fashion. Therefore, pinniped CTI may be critical for preventing the proteolytic degradation of immunoglobulins that are passively transferred from mother to young via colostrum and milk. Copyright © 2015 Elsevier B.V. All rights reserved.

Studies on the interactions between purified bovine caseins and alkaline-earth-metalions

PubMed Central

Dickson, I. R.; Perkins, D. J.

1971-01-01

1. Alkaline-earth-metal cations at low concentrations form soluble complexes with bovine caseins. The relative order of binding capacities is: Mg2+>Ca2+>Ba2+>Sr2+. 2. The cations interact with both free ionized carboxyl groups of aspartic acid and glutamic acid and with monoester phosphate groups covalently bound to serine and threonine; at low concentrations of the cations interactions are predominantly with the phosphate groups. 3. The order of binding capacities for purified components of the casein complex is: αs1-casein>β-casein>κ-casein. PMID:5166590

Susceptibility of the individual caseins in reconstituted skim milk to cross-linking by transglutaminase: influence of temperature, pH and mineral equilibria.

PubMed

Hinz, Katharina; Huppertz, Thom; Kelly, Alan L

2012-11-01

The susceptibility of total casein and the individual caseins in reconstituted skim milk to transglutaminase (TGase)-induced cross-linking was studied as a function of incubation temperature (5-40 °C), pH (5·0-7·0) and mineral addition. Within the ranges studied, the level of total casein cross-linked increased with increasing temperature, pH and concentration of added trisodium citrate, whereas adding calcium chloride had the opposite effect. These effects can be largely related to the effects of these parameters on TGase activity. In addition, the parameters were also found to influence the susceptibility of κ-casein, and to a lesser extent β-casein, to cross-linking, whereas the susceptibility of αs1-casein was not affected. The susceptibility of κ-casein to cross-linking increased with increasing temperature and calcium chloride addition, but decreased with increasing pH and citrate content, whereas the susceptibility of β-casein to TGase-induced cross-linking decreased with increasing temperature, but was not affected by other parameters. These findings highlight the fact that selection of environmental conditions during cross-linking can be applied to tailor the surface, and hence possibly colloidal stability, of casein micelles in TGase-treated milk.

Disruption and reassociation of casein micelles under high pressure: influence of milk serum composition and casein micelle concentration.

PubMed

Huppertz, Thom; de Kruif, Cornelis G

2006-08-09

In this study, factors influencing the disruption and aggregation of casein micelles during high-pressure (HP) treatment at 250 MPa for 40 min were studied in situ in serum protein-free casein micelle suspensions. In control milk, light transmission increased with treatment time for approximately 15 min, after which a progressive partial reversal of the HP-induced increase in light transmission occurred, indicating initial HP-induced disruption of casein micelles, followed by reformation of casein aggregates from micellar fragments. The extent of HP-induced micellar disruption was negatively correlated with the concentration of casein micelles, milk pH, and levels of added ethanol, calcium chloride, or sodium chloride and positively correlated with the level of added sodium phosphate. The reformation of casein aggregates during prolonged HP treatment did not occur when HP-induced disruption of casein micelles was limited (<60%) or very extensive (>95%) and was promoted by a low initial milk pH or added sodium phosphate, sodium chloride, or ethanol. On the basis of these findings, a mechanism for HP-induced disruption of casein micelles and subsequent aggregation of micellar fragments is proposed, in which the main element appears to be HP-induced solubilization of micellar calcium phosphate.

Dairy Attenuates Weight Gain to a Similar Extent as Exercise in Rats Fed a High-Fat, High-Sugar Diet.

PubMed

Trottier, Sarah K; MacPherson, Rebecca E K; Knuth, Carly M; Townsend, Logan K; Peppler, Willem T; Mikhaeil, John S; Leveille, Cam F; LeBlanc, Paul J; Shearer, Jane; Reimer, Raylene A; Wright, David C

2017-10-01

To compare the individual and combined effects of dairy and endurance exercise training in reducing weight gain and adiposity in a rodent model of diet-induced obesity. An 8-week feeding intervention of a high-fat, high-sugar diet was used to induce obesity in male Sprague-Dawley rats. Rats were then assigned to one of four groups for 6 weeks: (1) casein sedentary (casein-S), (2) casein exercise (casein-E), (3) dairy sedentary (dairy-S), and (4) dairy exercise (dairy-E). Rats were exercise trained by treadmill running 5 d/wk. Dairy-E prevented weight gain to a greater extent than either dairy or exercise alone. Adipose tissue and liver mass were reduced to a similar extent in dairy-S, casein-E, and dairy-E groups. Differences in weight gain were not explained by food intake or total energy expenditure. The total amount of lipid excreted was greater in the dairy-S compared to casein-S and dairy-E groups. This study provides evidence that dairy limits weight gain to a similar extent as exercise training and the combined effects are greater than either intervention alone. While exercise training reduces weight gain through increases in energy expenditure, dairy appears to increase lipid excretion in the feces. © 2017 The Obesity Society.

Grafting C8-C16 alkyl groups altered the self-assembly and curcumin -loading properties of sodium caseinate in water.

PubMed

Zhang, Yaqiong; Yang, Puyu; Yao, Fangyi; Liu, Jie; Yu, Liangli Lucy

2018-02-01

The data presented here are related to the research article entitled "Synthesis and characterization of alkylated caseinate, and its structure-curcumin loading property relationship in water" (Zhang et al., 2018) [1]. This data article reports the detailed spectra information for 1 H NMR, 13 C NMR and UPLC-Q-TOF MS of the N-succinimidyl fatty acid esters with various alkyl chain lengths (Cn-NHSs, n = 8, 12, 14 and 16). 1 H NMR, 13 C NMR and UPLC-Q-TOF MS spectra for C16-NHS are shown as an example. Then the stacked 1 H NMR spectra of the obtained alkylated caseinates (Cn-caseinates, n = 8, 12, 14 and 16) are provided. The surface hydrophobicity index (S 0 ) of Cn-caseinates with different substitution degrees (SD) of alkyl groups is shown. Additionally, Visual appearances for the formed aqueous dispersions of curcumin-loaded native caseinate (NaCas) and Cn-caseinates self-assemblies are shown. X-ray diffraction patterns of curcumin, C16-caseinate, its physical mixture and curcumin-loaded C16-caseinate self-assemblies are examined. The re-dispersibility and short-term storage stability of the curcumin-loaded NaCas and C16-caseinate self-assemblies are also studied.

Transglutaminase-treated conjugation of sodium caseinate and corn fiber gum hydrolysate: Interfacial and dilatational properties.

PubMed

Liu, Yan; Selig, Michael J; Yadav, Madhav P; Yin, Lijun; Abbaspourrad, Alireza

2018-05-01

This study compliments previous work where peroxidase was successfully used to crosslink corn fiber gum (CFG) with bovine serum albumin and improve CFG's emulsifying properties. Herein, an alternative type of enzyme, transglutaminase, was used to prepare conjugates of CFG and sodium caseinate. Additionally, the CFG was partially hydrolyzed by sulfuric acid and its crosslinking pattern with caseinate was evaluated. The interfacial crosslinking degree between caseinate and CFG increased after hydrolysis according to high performance size exclusion chromatography. The equilibrium interfacial tension of CFG hydrolysate-caseinate conjugate was lower than that of CFG-caseinate conjugate as the rearrangement rate of the CFG hydrolysate-caseinate conjugate was higher. The dilatational modulus of CFG hydrolysate decreased from that of CFG. Copyright © 2018 Elsevier Ltd. All rights reserved.

The membrane-associated form of α(s1)-casein interacts with cholesterol-rich detergent-resistant microdomains.

PubMed

Le Parc, Annabelle; Honvo Houéto, Edith; Pigat, Natascha; Chat, Sophie; Leonil, Joëlle; Chanat, Eric

2014-01-01

Caseins, the main milk proteins, interact with colloidal calcium phosphate to form the casein micelle. The mesostructure of this supramolecular assembly markedly influences its nutritional and technological functionalities. However, its detailed molecular organization and the cellular mechanisms involved in its biogenesis have been only partially established. There is a growing body of evidence to support the concept that α(s1)-casein takes center stage in casein micelle building and transport in the secretory pathway of mammary epithelial cells. Here we have investigated the membrane-associated form of α(s1)-casein in rat mammary epithelial cells. Using metabolic labelling we show that α(s1)-casein becomes associated with membranes at the level of the endoplasmic reticulum, with no subsequent increase at the level of the Golgi apparatus. From morphological and biochemical data, it appears that caseins are in a tight relationship with membranes throughout the secretory pathway. On the other hand, we have observed that the membrane-associated form of α(s1)-casein co-purified with detergent-resistant membranes. It was poorly solubilised by Tween 20, partially insoluble in Lubrol WX, and substantially insoluble in Triton X-100. Finally, we found that cholesterol depletion results in the release of the membrane-associated form of α(s1)-casein. These experiments reveal that the insolubility of α(s1)-casein reflects its partial association with a cholesterol-rich detergent-resistant microdomain. We propose that the membrane-associated form of α(s1)-casein interacts with the lipid microdomain, or lipid raft, that forms within the membranes of the endoplasmic reticulum, for efficient forward transport and sorting in the secretory pathway of mammary epithelial cells.

The Membrane-Associated Form of αs1-Casein Interacts with Cholesterol-Rich Detergent-Resistant Microdomains

PubMed Central

Le Parc, Annabelle; Honvo Houéto, Edith; Pigat, Natascha; Chat, Sophie; Leonil, Joëlle; Chanat, Eric

2014-01-01

Caseins, the main milk proteins, interact with colloidal calcium phosphate to form the casein micelle. The mesostructure of this supramolecular assembly markedly influences its nutritional and technological functionalities. However, its detailed molecular organization and the cellular mechanisms involved in its biogenesis have been only partially established. There is a growing body of evidence to support the concept that αs1-casein takes center stage in casein micelle building and transport in the secretory pathway of mammary epithelial cells. Here we have investigated the membrane-associated form of αs1-casein in rat mammary epithelial cells. Using metabolic labelling we show that αs1-casein becomes associated with membranes at the level of the endoplasmic reticulum, with no subsequent increase at the level of the Golgi apparatus. From morphological and biochemical data, it appears that caseins are in a tight relationship with membranes throughout the secretory pathway. On the other hand, we have observed that the membrane-associated form of αs1-casein co-purified with detergent-resistant membranes. It was poorly solubilised by Tween 20, partially insoluble in Lubrol WX, and substantially insoluble in Triton X-100. Finally, we found that cholesterol depletion results in the release of the membrane-associated form of αs1-casein. These experiments reveal that the insolubility of αs1-casein reflects its partial association with a cholesterol-rich detergent-resistant microdomain. We propose that the membrane-associated form of αs1-casein interacts with the lipid microdomain, or lipid raft, that forms within the membranes of the endoplasmic reticulum, for efficient forward transport and sorting in the secretory pathway of mammary epithelial cells. PMID:25549363

Casein kinase II is required for the spindle assembly checkpoint by regulating Mad2p in fission yeast

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shimada, Midori; Yamamoto, Ayumu; Murakami-Tonami, Yuko

2009-10-23

The spindle checkpoint is a surveillance mechanism that ensures the fidelity of chromosome segregation in mitosis. Here we show that fission yeast casein kinase II (CK2) is required for this checkpoint function. In the CK2 mutants mitosis occurs in the presence of a spindle defect, and the spindle checkpoint protein Mad2p fails to localize to unattached kinetochores. The CK2 mutants are sensitive to the microtubule depolymerising drug thiabendazole, which is counteracted by ectopic expression of mad2{sup +}. The level of Mad2p is low in the CK2 mutants. These results suggest that CK2 has a role in the spindle checkpoint bymore » regulating Mad2p.« less

Interfacial composition and stability of emulsions made with mixtures of commercial sodium caseinate and whey protein concentrate.

PubMed

Ye, Aiqian

2008-10-15

The interfacial composition and the stability of oil-in-water emulsion droplets (30% soya oil, pH 7.0) made with mixtures of sodium caseinate and whey protein concentrate (WPC) (1:1 by protein weight) at various total protein concentrations were examined. The average volume-surface diameter (d32) and the total surface protein concentration of emulsion droplets were similar to those of emulsions made with both sodium caseinate alone and WPC alone. Whey proteins were adsorbed in preference to caseins at low protein concentrations (<3%), whereas caseins were adsorbed in preference to whey proteins at high protein concentrations. The creaming stability of the emulsions decreased markedly as the total protein concentration of the system was increased above 2% (sodium caseinate >1%). This was attributed to depletion flocculation caused by the sodium caseinate in these emulsions. Whey proteins did not retard this instability in the emulsions made with mixtures of sodium caseinate and WPC. Copyright © 2008 Elsevier Ltd. All rights reserved.

Interaction of flavan-3-ol derivatives and different caseins is determined by more than proline content and number of proline repeats.

PubMed

Bohin, Maxime C; Vincken, Jean-Paul; Westphal, Adrie H; Tripp, Annelise M; Dekker, Peter; van der Hijden, Harry T W M; Gruppen, Harry

2014-09-01

Interactions of Type A and B flavan-3-ol dimers (procyanidins) and several monomeric flavan-3-ols, with α-casein and β-casein, were investigated. Binding affinities measured were related to the ligands structure, including several properties (e.g. intrinsic flexibility (number of rotatable bonds) and hydrophobicity), and to the amino-acid composition of the caseins. A monomeric flavan-3-ol esterified with gallic acid (EGCG) had a five to ten times higher affinity to caseins compared to the non-galloylated dimeric flavan-3-ols. In this case, the larger number of rotatable bonds in EGCG might be accountable for this difference. Comparing flavan-3-ol dimers, intrinsic flexibility did not consistently promote interactions, as procyanidin A1 displayed a higher affinity to α-casein than the supposedly more flexible B-type dimers investigated. Despite its higher content of proline, compared to α-casein, β-casein did not always have a higher affinity for the ligands investigated (e.g. no interaction with procyanidin A1 detected). These results suggest that more factors than proline content and the number of proline repeats govern phenolic-casein interactions. Copyright © 2014 Elsevier Ltd. All rights reserved.

Sequence of rat alpha- and gamma-casein mRNAs: evolutionary comparison of the calcium-dependent rat casein multigene family.

PubMed Central

Hobbs, A A; Rosen, J M

1982-01-01

The complete sequences of rat alpha- and gamma-casein mRNAs have been determined. The 1402-nucleotide alpha- and 864-nucleotide gamma-casein mRNAs both encode 15 amino acid signal peptides and mature proteins of 269 and 164 residues, respectively. Considerable homology between the 5' non-coding regions, and the regions encoding the signal peptides and the phosphorylation sites, in these mRNAs as compared to several other rodent casein mRNAs, was observed. Significant homology was also detected between rat alpha- and bovine alpha s1-casein. Comparison of the rodent and bovine sequences suggests that the caseins evolved at about the time of the appearance of the primitive mammals. This may have occurred by intragenic duplication of a nucleotide sequence encoding a primitive phosphorylation site, -(Ser)n-Glu-Glu-, and intergenic duplication resulting in the small casein multigene family. A unique feature of the rat alpha-casein sequence is an insertion in the coding region containing 10 repeated elements of 18 nucleotides each. This insertion appears to have occurred 7-12 million years ago, just prior to the divergence of rat and mouse. Images PMID:6298707

The effect of ultrasound on casein micelle integrity.

PubMed

Chandrapala, J; Martin, G J O; Zisu, B; Kentish, S E; Ashokkumar, M

2012-12-01

Samples of fresh skim milk, reconstituted micellar casein, and casein powder were sonicated at 20 kHz to investigate the effect of ultrasonication. For fresh skim milk, the average size of the remaining fat globules was reduced by approximately 10 nm after 60 min of sonication; however, the size of the casein micelles was determined to be unchanged. A small increase in soluble whey protein and a corresponding decrease in viscosity also occurred within the first few minutes of sonication, which could be attributed to the breakup of casein-whey protein aggregates. No measurable changes in free casein content could be detected in ultracentrifuged skim milk samples sonicated for up to 60 min. A small, temporary decrease in pH resulted from sonication; however, no measurable change in soluble calcium concentration was observed. Therefore, casein micelles in fresh skim milk were stable during the exposure to ultrasonication. Similar results were obtained for reconstituted micellar casein, whereas larger viscosity changes were observed as whey protein content was increased. Controlled application of ultrasound can be usefully applied to reverse process-induced protein aggregation without affecting the native state of casein micelles. Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Gluten-free and casein-free diets in the therapy of autism.

PubMed

Lange, Klaus W; Hauser, Joachim; Reissmann, Andreas

2015-11-01

The purpose of this study is to discuss the role of gluten-free and casein-free diets in the treatment of autism. In a recent UK survey, more than 80% of parents of children with autism spectrum disorder reported some kind of dietary intervention for their child (gluten-free and casein-free diet in 29%). When asked about the effects of the gluten-free and casein-free diet, 20-29% of the parents reported significant improvements on the autism spectrum disorder core dimensions. The findings of this study suggest additional effects of a gluten-free and casein-free diet on comorbid problems of autism such as gastrointestinal symptoms, concentration, and attention. The findings of another recent investigation suggested that age and certain urine compounds may predict the response of autism symptoms to a gluten-free and casein-free diet. Although these results need to be replicated, they highlight the importance of patient subgroup analysis. Intervention trials evaluating the effects of a gluten-free and casein-free diet on autistic symptoms have so far been contradictory and inconclusive. Most investigations assessing the efficacy of a gluten-free and casein-free diet in the treatment of autism are seriously flawed. The evidence to support the therapeutic value of this diet is limited and weak. A gluten-free and casein-free diet should only be administered if an allergy or intolerance to nutritional gluten or casein is diagnosed.

Surface-Bound Casein Modulates the Adsorption and Activity of Kinesin on SiO2 Surfaces

PubMed Central

Ozeki, Tomomitsu; Verma, Vivek; Uppalapati, Maruti; Suzuki, Yukiko; Nakamura, Mikihiko; Catchmark, Jeffrey M.; Hancock, William O.

2009-01-01

Abstract Conventional kinesin is routinely adsorbed to hydrophilic surfaces such as SiO2. Pretreatment of surfaces with casein has become the standard protocol for achieving optimal kinesin activity, but the mechanism by which casein enhances kinesin surface adsorption and function is poorly understood. We used quartz crystal microbalance measurements and microtubule gliding assays to uncover the role that casein plays in enhancing the activity of surface-adsorbed kinesin. On SiO2 surfaces, casein adsorbs as both a tightly bound monolayer and a reversibly bound second layer that has a dissociation constant of 500 nM and can be desorbed by washing with casein-free buffer. Experiments using truncated kinesins demonstrate that in the presence of soluble casein, kinesin tails bind well to the surface, whereas kinesin head binding is blocked. Removing soluble casein reverses these binding profiles. Surprisingly, reversibly bound casein plays only a moderate role during kinesin adsorption, but it significantly enhances kinesin activity when surface-adsorbed motors are interacting with microtubules. These results point to a model in which a dynamic casein bilayer prevents reversible association of the heads with the surface and enhances association of the kinesin tail with the surface. Understanding protein-surface interactions in this model system should provide a framework for engineering surfaces for functional adsorption of other motor proteins and surface-active enzymes. PMID:19383474

Anticancer activity of cow, sheep, goat, mare, donkey and camel milks and their caseins and whey proteins and in silico comparison of the caseins.

PubMed

Shariatikia, Malihe; Behbahani, Mandana; Mohabatkar, Hassan

2017-06-01

The present investigation was carried out to evaluate anticancer activity of cow, goat, sheep, mare, donkey and camel milks and their casein and whey proteins against MCF7 cell line. The structure-based properties of the casein proteins were also investigated, using bioinformatics tools to find explanation for their antitumor activities. The effect of different milks and their casein and whey proteins on MCF7 proliferation was measured using MTT assay at different concentrations (0.5, 1 and 2 mg/ml). The results showed that mare, donkey, cow and camel milks and their casein and whey proteins have potent cytotoxic activity against MCF7 cells in a dose dependent manner while sheep and goat milks and their proteins did not reveal any cytotoxic activity. The in silico results demonstrated that mare, donkey and camel caseins had highest positive and negative charges. The secondary structure prediction indicated that mare and donkey caseins had the maximum percentage of α helix and camel casein had the highest percentage of extended strand. This study suggests that there is a striking correlation between anti-cancer activity of milk caseins and their physicochemical properties such as alpha helix structure and positive and negative charges. In conclusion, the results indicated that mare, camel and donkey milks might be good candidates against breast cancer cells.

Antiviral activity of casein and αs2 casein hydrolysates against the infectious haematopoietic necrosis virus, a rhabdovirus from salmonid fish.

PubMed

Rodríguez Saint-Jean, S; De las Heras, A; Carrillo, W; Recio, I; Ortiz-Delgado, J B; Ramos, M; Gomez-Ruiz, J A; Sarasquete, C; Pérez-Prieto, S I

2013-05-01

Salmonid fish viruses, such as infectious haematopoietic necrosis virus (IHNV), are responsible for serious losses in the rainbow trout and salmon-farming industries, and they have been the subject of intense research in the field of aquaculture. Thus, the aim of this work is to study the antiviral effect of milk-derived proteins as bovine caseins or casein-derived peptides at different stages during the course of IHNV infection. The results indicate that the 3-h fraction of casein and α(S2) -casein hydrolysates reduced the yield of infectious IHNV in a dose-dependent manner and impaired the production of IHNV-specific antigens. Hydrolysates of total casein and α(S2) -casein target the initial and later stages of viral infection, as demonstrated by the reduction in the infective titre observed throughout multiple stages and cycles. In vivo, more than 50% protection was observed in the casein-treated fish, and the kidney sections exhibited none of the histopathological characteristics of IHNV infection. The active fractions from casein were identified, as well as one of the individual IHNV-inhibiting peptides. Further studies will be required to determine which other peptides possess this activity. These findings provide a basis for future investigations on the efficacy of these compounds in treating other viral diseases in farmed fish and to elucidate the underlying molecular mechanisms of action. However, the present results provide convincing evidence in support of a role for several milk casein fractions as suitable candidates to prevent and treat some fish viral infections. © 2012 Blackwell Publishing Ltd.

Molecular characterization and phylogenetic analysis of a yak (Bos grunniens) κ-casein cDNA from lactating mammary gland.

PubMed

Bai, W L; Yin, R H; Dou, Q L; Jiang, W Q; Zhao, S J; Ma, Z J; Luo, G B; Zhao, Z H

2011-04-01

κ-Casein is one of the major proteins in the milk of mammals. It plays an important role in determining the size and specific function of milk micelles. We have previously identified and characterized a genetic variant of yak κ-casein by evaluating genomic DNA. Here, we isolate and characterize a yak κ-casein cDNA harboring the full-length open reading frame (ORF) from lactating mammary gland. Total RNA was extracted from mammary tissue of lactating female yak, and the κ-casein cDNA were synthesized by RT-PCR technique, then cloned and sequenced. The obtained cDNA of 660-bp contained an ORF sufficient to encode the entire amino acid sequence of κ-casein precursor protein consisting of 190 amino acids with a signal peptide of 21 amino acids. Yak κ-casein has a predicted molecular mass of 19,006.588 Da with a calculated isoelectric point of 7.245. Compared with the corresponding sequences in GenBank of cattle, buffalo, sheep, goat, Arabian camel, horse, and rabbit, yak κ-casein sequence had identity of 64.76-98.78% in cDNA, and identity of 44.79-98.42% and similarity of 53.65-98.42% in deduced amino acids, revealing a high homology with the other livestock species. Based on κ-casein cDNA sequences, the phylogenetic analysis indicated that yak κ-casein had a close relationship with that of cattle. This work might be useful in the genetic engineering researches for yak κ-casein.

Magnetic bead and gold nanoparticle probes based immunoassay for β-casein detection in bovine milk samples.

PubMed

Li, Y S; Meng, X Y; Zhou, Y; Zhang, Y Y; Meng, X M; Yang, L; Hu, P; Lu, S Y; Ren, H L; Liu, Z S; Wang, X R

2015-04-15

In this work, a double-probe based immunoassay was developed for rapid and sensitive determination of β-casein in bovine milk samples. In the method, magnetic beads (MBs), employed as supports for the immobilization of anti-β-casein polyclonal antibody (PAb), were used as the capture probe. Colloidal gold nanoparticles (AuNPs), employed as a bridge for loading anti-β-casein monoclonal antibody (McAb) and horseradish peroxidase (HRP), were used as the amplification probe. The presence of β-casein causes the sandwich structures of MBs-PAb-β-casein-McAb-AuNPs through the interaction between β-casein and the anti-β-casein antibodies. The HRP, used as an enzymatic-amplified tracer, can catalytically oxidize the substrate 3,3',5,5'-tetramethylbenzidine (TMB), generating optical signals that are proportional to the quantity of β-casein. The linear range of the immunoassay was from 6.5 to 1520ngmL(-1). The limit of detection (LOD) was 4.8ngmL(-1) which was 700 times lower than that of MBs-antibody-HRP based immunoassay and 6-7 times lower than that from the microplate-antibody-HRP based assay. The recoveries of β-casein from bovine milk samples were from 95.0% to 104.3% that had a good correlation coefficient (R(2)=0.9956) with those obtained by an official standard Kjeldahl method. For higher sensitivity, simple sample pretreatment and shorter time requirement of the antigen-antibody reaction, the developed immunoassay demonstrated the viability for detection of β-casein in bovine milk samples. Copyright © 2014. Published by Elsevier B.V.

Preparation and bioactive properties of chitosan and casein phosphopeptides composite coatings for orthopedic implants.

PubMed

Qin, Liguo; Dong, Huanhuan; Mu, Ziqing; Zhang, Yali; Dong, Guangneng

2015-11-20

Using the layer-by-layer deposition method, functional chitosan/casein phospopeptides (CS/CPP) composite coatings were produced on Co-Cr-Mo alloy. The CS/CPP composite coatings had the dendritic topography, and were quite hydrophilic. Zeta potential measurements showed the composite coatings were negative charged at neural pH. XPS results indicated that the CS/CPP composite coatings were covalently bond to the substrate. When MC3T3-E1 cells were seeded on the CS/CPP composite coatings, no cytotoxicity was observed. The bone morphogenetic protein-2 (BMP-2) mRNA expression was significantly up-regulated in MC3T3-E1 cells cultured on the composite coatings and it was twice as much as that of cells cultured on the bare substrate. The expression of osteoprotegerin (OPG) mRNA and the ratio of OPG/receptor activator of nuclear factor-κB ligand (RNAKL) mRNA were increased 5-fold and 55-fold, respectively. These results suggested the CS/CPP composite coatings may have potential application in cobalt matrix orthopaedic implants. Copyright © 2015 Elsevier Ltd. All rights reserved.

Effect of polymorphisms in the CSN3 (κ-casein) gene on milk production traits in Chinese Holstein Cattle.

PubMed

Alim, M A; Dong, T; Xie, Y; Wu, X P; Zhang, Yi; Zhang, Shengli; Sun, D X

2014-11-01

This study was designed to evaluate significant associations between single nucleotide polymorphisms (SNPs) and milk composition and milk production traits in Chinese Holstein cows. Six SNPs were identified in the κ-casein gene using pooled DNA sequencing. The identified SNPs were genotyped by Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) methods from 507 individuals. Out of six, we identified three non-synonymous SNPs (g.10888T>C, g.10924C>A and g.10944A>G) that changed in the protein product. SIFT (Sorting_Intolerant_From_Tolerant) prediction score (0.01) demonstrated that protein changed Isoleucine > Threonine (g.10888T>C) will affect the phenotypes. Significant associations between identified SNPs and three yield traits (milk, protein and fat) and two composition traits (fat and protein percentages) were found whereas it did not reach significance for fat percentage in haplotypes association. Importantly, the significant SNPs in our results showed a large proportion of the phenotypic variation of milk protein yield and concentration. Our results suggest that CSN3 is an important candidate gene that influences milk production traits, and identified polymorphisms and haplotypes could be used as a genetic marker in programs of marker-assisted selection for the genetic improvement of milk production traits in dairy cattle.

21 CFR 520.1157 - Iodinated casein tablets.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Iodinated casein tablets. 520.1157 Section 520...) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS ORAL DOSAGE FORM NEW ANIMAL DRUGS § 520.1157 Iodinated casein tablets. (a) Specifications. Each 1-gram tablet contains 25 milligrams of iodinated casein. (b) Sponsor...

21 CFR 520.1157 - Iodinated casein tablets.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Iodinated casein tablets. 520.1157 Section 520...) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS ORAL DOSAGE FORM NEW ANIMAL DRUGS § 520.1157 Iodinated casein tablets. (a) Specifications. Each 1-gram tablet contains 25 milligrams of iodinated casein. (b) Sponsor...

21 CFR 182.1748 - Sodium caseinate.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Sodium caseinate. 182.1748 Section 182.1748 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR... Substances § 182.1748 Sodium caseinate. (a) Product. Sodium caseinate. (b) Conditions of use. This substance...

21 CFR 182.1748 - Sodium caseinate.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Sodium caseinate. 182.1748 Section 182.1748 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR... Substances § 182.1748 Sodium caseinate. (a) Product. Sodium caseinate. (b) Conditions of use. This substance...

21 CFR 582.1748 - Sodium caseinate.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Sodium caseinate. 582.1748 Section 582.1748 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Additives § 582.1748 Sodium caseinate. (a) Product. Sodium caseinate. (b) Conditions of use. This substance...

21 CFR 582.1748 - Sodium caseinate.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Sodium caseinate. 582.1748 Section 582.1748 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Additives § 582.1748 Sodium caseinate. (a) Product. Sodium caseinate. (b) Conditions of use. This substance...

21 CFR 182.1748 - Sodium caseinate.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Sodium caseinate. 182.1748 Section 182.1748 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR... Substances § 182.1748 Sodium caseinate. (a) Product. Sodium caseinate. (b) Conditions of use. This substance...

21 CFR 182.1748 - Sodium caseinate.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Sodium caseinate. 182.1748 Section 182.1748 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR... Substances § 182.1748 Sodium caseinate. (a) Product. Sodium caseinate. (b) Conditions of use. This substance...

21 CFR 582.1748 - Sodium caseinate.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Sodium caseinate. 582.1748 Section 582.1748 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Additives § 582.1748 Sodium caseinate. (a) Product. Sodium caseinate. (b) Conditions of use. This substance...

21 CFR 182.1748 - Sodium caseinate.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Sodium caseinate. 182.1748 Section 182.1748 Food... GENERALLY RECOGNIZED AS SAFE Multiple Purpose GRAS Food Substances § 182.1748 Sodium caseinate. (a) Product. Sodium caseinate. (b) Conditions of use. This substance is generally recognized as safe when used in...

21 CFR 582.1748 - Sodium caseinate.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Sodium caseinate. 582.1748 Section 582.1748 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Additives § 582.1748 Sodium caseinate. (a) Product. Sodium caseinate. (b) Conditions of use. This substance...

21 CFR 582.1748 - Sodium caseinate.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Sodium caseinate. 582.1748 Section 582.1748 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Additives § 582.1748 Sodium caseinate. (a) Product. Sodium caseinate. (b) Conditions of use. This substance...

Casein infusion rate influences feed intake differently depending on metabolizable protein balance in dairy cows: A multilevel meta-analysis.

PubMed

Martineau, R; Ouellet, D R; Kebreab, E; Lapierre, H

2016-04-01

The effects of casein infusion have been investigated extensively in ruminant species. Its effect on responses in dry matter intake (DMI) has been reviewed and indicated no significant effect. The literature reviewed in the current meta-analysis is more extensive and limited to dairy cows fed ad libitum. A total of 51 studies were included in the meta-analysis and data were fitted to a multilevel model adjusting for the correlated nature of some studies. The effect size was the mean difference calculated by subtracting the means for the control from the casein-infused group. Overall, casein infusion [average of 333 g of dry matter (DM)/d; range: 91 to 1,092 g of DM/d] tended to increase responses in DMI by 0.18 kg/d (n=48 studies; 3 outliers). However, an interaction was observed between the casein infusion rate (IR) and the initial metabolizable protein (MP) balance [i.e., supply minus requirements (NRC, 2001)]. When control cows were in negative MP balance (n=27 studies), responses in DMI averaged 0.28 kg/d at mean MP balance (-264 g/d) and casein IR (336 g/d), and a 100g/d increment in the casein IR from its mean increased further responses by 0.14 kg/d (MP balance being constant), compared with cows not infused with casein. In contrast, when control cows were in positive MP balance (n=22 studies; 2 outliers), responses in DMI averaged -0.20 kg/d at mean casein IR (339 g/d), and a 100g/d increment in the casein IR from its mean further decreased responses by 0.33 kg/d, compared with cows not infused with casein. Responses in milk true protein yield at mean casein IR were greater (109 vs. 65 g/d) for cows in negative vs. positive MP balance, respectively, and the influence of the casein IR on responses was significant only for cows in negative MP balance. A 100g/d increment in the casein IR from its mean increased further responses in milk true protein yield by 25 g/d, compared with cows not infused with casein. Responses in blood urea concentration increased in casein studies (+0.59 mM) and the influence of the casein IR was greatest for cows in positive MP balance (0.26 vs. 0.11 mM per 100g/d increment). Responses in DMI were also correlated negatively with responses in blood urea concentration only for cows in positive MP balance. Together, these results suggest an association between satiety and deamination and oxidation of AA supplied in excess of requirements for cows in positive MP balance. Therefore, casein stimulated appetite in cows fed MP-deficient diets possibly via the supply of orexigenic AA or through a pull effect in response to an increased metabolic demand. Conversely, casein induced satiety in cows fed diets supplying MP in excess of requirements. Not precluding other factors involved in satiety (e.g., insulin, gut peptides), casein could have increased the supply of AA (e.g., Ser, Thr, Tyr), which might depress appetite at the brain level or increase the deamination and the oxidation of AA in oversupply in agreement with the hepatic oxidation theory. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Milk protein polymorphisms in cattle (Bos indicus), mithun (Bos frontalis) and yak (Bos grunniens) breeds and their hybrids indigenous to Bhutan.

PubMed

Dorji, Tashi; Namikawa, Takao; Mannen, Hideyuki; Kawamoto, Yoshi

2010-10-01

In the current study, milk protein variation was examined in cattle (Bos indicus), mithun (Bos frontalis), yak (Bos grunniens) and their hybrid populations in Bhutan to estimate genetic variability, conduct genetic characterization and assess the possibility of gene flow between mithun and cattle. Isoelectric focusing of 372 milk samples from 11 populations detected four molecular types of β-lactoglobulin (A, B, E and M), five molecular types of α(S1) -casein (A, B, C, E and X) and three molecular types of k-casein (A, B and X). Mithun and yak shared alleles but were found to exhibit different allele frequencies for the proteins studied. The degree of genetic variability within populations was measured by average heterozygosity and ranged from 24-40% in cattle, 26% for yak and 33% for mithun. We also resolved the traditional mithun and cattle hybridization system via principal component analysis. Our results suggested secondary introgression of mithun genes to the village Thrabum population, and a close genetic relationship between Bhutanese indigenous cattle and Indian cattle. © 2010 The Authors; Journal compilation © 2010 Japanese Society of Animal Science.

Phosphorylation by casein kinase 2 facilitates rRNA gene transcription by promoting dissociation of TIF-IA from elongating RNA polymerase I.

PubMed

Bierhoff, Holger; Dundr, Miroslav; Michels, Annemieke A; Grummt, Ingrid

2008-08-01

The protein kinase casein kinase 2 (CK2) phosphorylates different components of the RNA polymerase I (Pol I) transcription machinery and exerts a positive effect on rRNA gene (rDNA) transcription. Here we show that CK2 phosphorylates the transcription initiation factor TIF-IA at serines 170 and 172 (Ser170/172), and this phosphorylation triggers the release of TIF-IA from Pol I after transcription initiation. Inhibition of Ser170/172 phosphorylation or covalent tethering of TIF-IA to the RPA43 subunit of Pol I inhibits rDNA transcription, leading to perturbation of nucleolar structure and cell cycle arrest. Fluorescence recovery after photobleaching and chromatin immunoprecipitation experiments demonstrate that dissociation of TIF-IA from Pol I is a prerequisite for proper transcription elongation. In support of phosphorylation of TIF-IA switching from the initiation into the elongation phase, dephosphorylation of Ser170/172 by FCP1 facilitates the reassociation of TIF-IA with Pol I, allowing a new round of rDNA transcription. The results reveal a mechanism by which the functional interplay between CK2 and FCP1 sustains multiple rounds of Pol I transcription.

The plasminogen activator system in the ovine placentome during late gestation and stage-two of parturition.

PubMed

McNeel, Anthony K; Cushman, Robert A; Vallet, Jeffrey L

2013-06-01

The process of placental separation is not completely understood. In domestic animals, especially cattle, it is important that expulsion of the fetal membranes takes place in a timely manner in order to achieve maximal reproductive efficiency. The activity of the matrix-metalloprotease (MMP) family of proteases is known to be reduced in placentomes from cases of retained placenta. Members of the MMP family are known to be activated by the plasminogen activator (PA) family of proteases. We hypothesized that the expression and activity of the PA family increase in the cotyledon and/or caruncle as parturition approaches, with maximal expression and activity at parturition. To test this hypothesis, we performed reverse-transcriptase quantitative PCR and plasminogen-casein zymography to detect the presence and activity of PA family members in the placentome leading up to and during parturition in spontaneous and dexamethasone-induced parturient ewes. The results from our experiments indicated that serine proteases inhibitor E1 (SERPINE1) mRNA abundance in the cotyledon was different between treatment groups (P = 0.0002). In the caruncle, gene expression for plasminogen activator urokinase-type (PLAU) was different (P = 0.0154), and there was a strong trend for differences in SERPINE1 expression (P = 0.0565). These results demonstrate that expression of the PA system in the placentome changes from late pregnancy to parturition, and the presence or activity of these enzymes may occur after fetal expulsion.

Effects of Surface Passivation on Gliding Motility Assays

DTIC Science & Technology

2011-06-03

alpha and beta, is structurally very different. It is a glycoprotein and is thought to stabilize the casein micelle [20,44,45] by sterically hindering...the aggregation of too many casein sub- micelles . It did not support motility in a very consistent manner as can be seen from Figure 1C&D. As was the...between alpha and whole casein was not due to impurities but rather that it was due to how kinesin was supported by the casein micelles or, how casein

Casein kinase 2 inhibition impairs spontaneous and oxytocin-induced contractions in late pregnant mouse uterus.

PubMed

Suhas, K S; Parida, Subhashree; Gokul, Chandrasekaran; Srivastava, Vivek; Prakash, E; Chauhan, Sakshi; Singh, Thakur Uttam; Panigrahi, Manjit; Telang, Avinash G; Mishra, Santosh K

2018-05-01

What is the central question of this study? Does the inhibition of the protein kinase casein kinase 2 (CK2) alter the uterine contractility? What is the main finding and its importance? Inhibition of CK2 impaired the spontaneous and oxytocin-induced contractility in late pregnant mouse uterus. This finding suggests that CK2 is a novel pathway mediating oxytocin-induced contractility in the uterus and thus opens up the possibility for this class of drugs to be developed as a new class of tocolytics. The protein kinase casein kinase 2 (CK2) is a ubiquitously expressed serine or threonine kinase known to phosphorylate a number of substrates. The aim of this study was to assess the effect of CK2 inhibition on spontaneous and oxytocin-induced uterine contractions in 19 day pregnant mice. The CK2 inhibitor CX-4945 elicited a concentration-dependent relaxation in late pregnant mouse uterus. CX-4945 and another selective CK2 inhibitor, apigenin, also inhibited the oxytocin-induced contractile response in late pregnant uterine tissue. Apigenin also blunted the prostaglandin F 2α response, but CX-4945 did not. Casein kinase 2 was located in the lipid raft fractions of the cell membrane, and disruption of lipid rafts was found to reverse its effect. The results of the present study suggest that CK2, located in lipid rafts of the cell membrane, is an active regulator of spontaneous and oxytocin-induced uterine contractions in the late pregnant mouse. © 2018 The Authors. Experimental Physiology © 2018 The Physiological Society.

Restricted growth of U-type infectious haematopoietic necrosis virus (IHNV) in rainbow trout cells may be linked to casein kinase II activity

USGS Publications Warehouse

Park, J.-W.; Moon, C.H.; Harmache, A.; Wargo, A.R.; Purcell, M.K.; Bremont, M.; Kurath, G.

2011-01-01

Previously, we demonstrated that a representative M genogroup type strain of infectious haematopoietic necrosis virus (IHNV) from rainbow trout grows well in rainbow trout-derived RTG-2 cells, but a U genogroup type strain from sockeye salmon has restricted growth, associated with reduced genome replication and mRNA transcription. Here, we analysed further the mechanisms for this growth restriction of U-type IHNV in RTG-2 cells, using strategies that assessed differences in viral genes, host immune regulation and phosphorylation. To determine whether the viral glycoprotein (G) or non-virion (NV) protein was responsible for the growth restriction, four recombinant IHNV viruses were generated in which the G gene of an infectious IHNV clone was replaced by the G gene of U- or M-type IHNV and the NV gene was replaced by NV of U- or M-type IHNV. There was no significant difference in the growth of these recombinants in RTG-2 cells, indicating that G and NV proteins are not major factors responsible for the differential growth of the U- and M-type strains. Poly I:C pretreatment of RTG-2 cells suppressed the growth of both U- and M-type IHNV, although the M virus continued to replicate at a reduced level. Both viruses induced type 1 interferon (IFN1) and the IFN1 stimulated gene Mx1, but the expression levels in M-infected cells were significantly higher than in U-infected cells and an inhibitor of the IFN1-inducible protein kinase PKR, 2-aminopurine (2-AP), did not affect the growth of U- or M-type IHNV in RTG-2 cells. These data did not indicate a role for the IFN1 system in the restricted growth of U-type IHNV in RTG-2 cells. Prediction of kinase-specific phosphorylation sites in the viral phosphoprotein (P) using the NetPhosK program revealed differences between U- and M-type P genes at five phosphorylation sites. Pretreatment of RTG-2 cells with a PKC inhibitor or a p38MAPK inhibitor did not affect the growth of the U- and M-type viruses. However, 100 μm of the casein kinase II (CKII) inhibitor, 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB), reduced the titre of the U type 8.3-fold at 24 h post-infection. In contrast, 100 μm of the CKII inhibitor reduced the titre of the M type only 1.3-fold at 48 h post-infection. Our data suggest that the different growth of U- and M-type IHNV in RTG-2 cells may be linked to a differential requirement for cellular protein kinases such as CKII for their growth.

Sustained activation of STAT5 is essential for chromatin remodeling and maintenance of mammary-specific function

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Ren; Nelson, Celeste M.; Muschler, John L.

2009-06-03

Epithelial cells, once dissociated and placed in two-dimensional (2D) cultures, rapidly lose tissue-specific functions. We showed previously that in addition to prolactin, signaling by laminin-111 was necessary to restore functional differentiation of mammary epithelia. Here, we elucidate two additional aspects of laminin-111 action. We show that in 2D cultures, the prolactin receptor is basolaterally localized and physically segregated from its apically placed ligand. Detachment of the cells exposes the receptor to ligation by prolactin leading to signal transducers and activators of transcription protein 5 (STAT5) activation, but only transiently and not sufficiently for induction of milk protein expression. We showmore » that laminin-111 reorganizes mammary cells into polarized acini, allowing both the exposure of the prolactin receptor and sustained activation of STAT5. The use of constitutively active STAT5 constructs showed that the latter is necessary and sufficient for chromatin reorganization and {beta}-casein transcription. These results underscore the crucial role of continuous laminin signaling and polarized tissue architecture in maintenance of transcription factor activation, chromatin organization, and tissue-specific gene expression.« less

CASEIN KINASE-MEDIATED PHOSPHORYLATION OF SERINE 839 IS NECESSARY FOR BASOLATERAL LOCALIZATION OF THE Ca2+-ACTIVATED NON-SELECTIVE CATION CHANNEL TRPM4

PubMed Central

Cerda, Oscar; Cáceres, Mónica; Park, Kang-Sik; Leiva-Salcedo, Elías; Romero, Aníbal; Varela, Diego

2014-01-01

TRPM4 is a Ca2+-activated non-selective cation channel expressed in a wide range of human tissues. TRPM4 participates in a variety of physiological processes such as T cell activation, myogenic vasoconstriction and allergic reactions. TRPM4 Ca2+ sensitivity is enhanced by calmodulin (CaM) and phosphathydilinositol 4, 5-biphosphate (PI(4,5)P2) binding, as well as, under certain conditions, PKC activation. However, information as to the mechanisms of modulation of this channel remain unknown, including direct identification of phosphorylation sites on TRPM4 and their role in channel features. Here, we use mass-spectrometric-based proteomic approaches (immunoprecipitation and tandem mass spectrometry), to unambiguously identify S839 as a phosphorylation site present on human TRPM4 expressed in a human cell line. Site-directed mutagenesis employing a serine to alanine mutation to eliminate phosphorylation, and a phospho-mimetic aspartate mutation, as well as biochemical and immunocytochemical experiments, revealed a role for S839 phosphorylation in the basolateral expression of TRPM4 channels in epithelial cells. Moreover, we demonstrated that casein kinase 1 (CK1) phosphorylates S839 and is responsible for the basolateral localization of TRPM4. PMID:25231975

Casein kinase-mediated phosphorylation of serine 839 is necessary for basolateral localization of the Ca²⁺-activated non-selective cation channel TRPM4.

PubMed

Cerda, Oscar; Cáceres, Mónica; Park, Kang-Sik; Leiva-Salcedo, Elías; Romero, Aníbal; Varela, Diego; Trimmer, James S; Stutzin, Andrés

2015-08-01

Transient receptor potential melastatin-like 4 (TRPM4) is a Ca(2+)-activated non-selective cation channel expressed in a wide range of human tissues. TRPM4 participates in a variety of physiological processes such as T cell activation, myogenic vasoconstriction, and allergic reactions. TRPM4 Ca(2+) sensitivity is enhanced by calmodulin (CaM) and phosphathydilinositol 4, 5-bisphosphate (PI(4,5)P2) binding, as well as, under certain conditions, PKC activation. However, information as to the mechanisms of modulation of this channel remains unknown, including direct identification of phosphorylation sites on TRPM4 and their role in channel features. Here, we use mass-spectrometric-based proteomic approaches (immunoprecipitation and tandem mass spectrometry) to unambiguously identify S839 as a phosphorylation site present on human TRPM4 expressed in a human cell line. Site-directed mutagenesis employing a serine to alanine mutation to eliminate phosphorylation, and a phospho-mimetic aspartate mutation, as well as biochemical and immunocytochemical experiments, revealed a role for S839 phosphorylation in the basolateral expression of TRPM4 channels in epithelial cells. Moreover, we demonstrated that casein kinase 1 (CK1) phosphorylates S839 and is responsible for the basolateral localization of TRPM4.

Use of sunlight to partially detoxify groundnut (peanut) cake flour and casein contaminated with aflatoxin B1.

PubMed

Shantha, T; Murthy, V S

1981-03-01

Sunlight destroyed 83 and 50% of the toxin added to casein and groundnut cake flour, respectively. Equilibrium dialysis revealed that both casein and groundnut protein bind aflatoxin but the toxin bound to casein appeared more photo-labile than that bound to groundnut protein.

Short communication: separation and quantification of caseins and casein macropeptide using ion-exchange chromatography.

PubMed

Holland, B; Rahimi Yazdi, S; Ion Titapiccolo, G; Corredig, M

2010-03-01

The aim of this work was to improve an existing method to separate and quantify the 4 major caseins from milk samples (i.e., containing whey proteins) using ion-exchange chromatography. The separation process was carried out using a mini-preparative cation exchange column (1 or 5mL of column volume), using urea acetate as elution buffer at pH 3.5 with a NaCl gradient. All 4 major caseins were separated, and the purity of each peak was assessed using sodium dodecyl sulfate-PAGE. Purified casein fractions were also added to raw milk to confirm their elution volumes. The quantification was carried out using purified caseins in buffer as well as added directly to fresh skim milk. This method can also be employed to determine the decrease in kappa-casein and the release of the casein-macropeptide during enzymatic hydrolysis using rennet. In this case, the main advantage of using this method is the lack of organic solvents compared with the conventional method for separation of macropeptide (using reversed phase HPLC).

Reassociation of dissociated caseins upon acidification of heated pH-adjusted skim milk.

PubMed

Anema, Skelte G; Li, Yuming

2015-05-01

Milk was heated at different pH (pH 6.5-7.1) and temperatures (20-120 °C/10 min). This resulted in different levels of casein and denatured whey proteins to be distributed between the colloidal and serum phases. The milks were subsequently acidified and the distribution of protein between colloidal and serum was monitored at different pH. On acidification to pH 5.4, the serum phase caseins and denatured whey proteins partially reassociated with the caseins, although a complex behaviour was observed at ∼ pH 5.4 where additional casein dissociation occurred in some samples. At pH below 5.4 the caseins and denatured whey proteins rapidly aggregated. No separate aggregation of κ-casein/denatured whey protein complexes or κ-casein depleted micelles was observed. The earlier gelation of milks heated at higher pH was likely to be due to the destabilisation of the entire milk protein system rather than a preferential aggregation of the serum phase proteins. Copyright © 2014 Elsevier Ltd. All rights reserved.

Dietary whey proteins shield murine cecal microbiota from extensive disarray caused by a high-fat diet.

PubMed

Monteiro, Naice E S; Roquetto, Aline R; de Pace, Fernanda; Moura, Carolina S; Santos, Andrey Dos; Yamada, Aureo T; Saad, Mário José A; Amaya-Farfan, Jaime

2016-07-01

High-fat diets are used to induce adverse alterations in the intestinal microbiota, or dysbiosis, generalized inflammation and metabolic stress, which ultimately may lead to obesity. The influence of dietary whey proteins, whether intact or hydrolyzed, has been reported to improve glucose homeostasis and reduce stress. Therefore, the purpose of this work was to test if dietary milk-whey proteins, both in the intact form and hydrolyzed, could have an effect on the compositional changes of the cecal microbiota that can be induced in mice when receiving a high-fat diet in combination with the standard casein. Male C57BL/6 mice were fed a control casein diet (AIN 93-G); high-fat-casein (HFCAS); high-fat-whey protein concentrate (HFWPC) and high-fat whey-protein hydrolysate (HFWPH) for 9weeks. The intestinal microbiota composition was analyzed by 16S-rRNA of the invariant (V1-V3) gene, potentially endotoxemic lipopolysaccharide (LPS) release was determined colorimetrically, and liver fat infiltration assessed by light microscopy. The high-fat diet proved to induce dysbiosis in the animals by inverting the dominance of the phylum Firmicutes over Bacteroidetes, promoted the increase of LPS and resulted in liver fat infiltration. The whey proteins, whether intact or hydrolyzed, resisted the installation of dysbiosis, prevented the surge of circulating LPS and prevented fat infiltration in the liver. It is concluded that dietary whey proteins exert metabolic actions that tend to preserve the normal microbiota profile, while mitigating liver fat deposition in mice consuming a high-fat diet for nine weeks. Such beneficial effects were not seen when casein was the dietary protein. The hydrolyzed whey protein still differed from the normal whey protein by selectively protecting the Bacteroidetes phylum. Copyright © 2016 Elsevier Ltd. All rights reserved.

Proteomic analysis and cross species comparison of casein fractions from the milk of dairy animals

PubMed Central

Wang, Xiaxia; Zhao, Xiaowei; Huang, Dongwei; Pan, Xiaocheng; Qi, Yunxia; Yang, Yongxin; Zhao, Huiling; Cheng, Guanglong

2017-01-01

Casein micelles contribute to the physicochemical properties of milk and may also influence its functionality. At present, however, there is an incomplete understanding of the casein micelle associated proteins and its diversity among the milk obtained from different species. Therefore, milk samples were collected from seven dairy animals groups, casein fractions were prepared by ultracentrifugation and their constituent proteins were identified by liquid chromatography tandem mass spectrometry. A total of 193 distinct proteins were identified among all the casein micelle preparations. Protein interaction analysis indicated that caseins could interact with major whey proteins, including β-lactoglobulin, α-lactalbumin, lactoferrin, and serum albumin, and then whey proteins interacted with other proteins. Pathway analysis found that the peroxisome proliferator-activated receptor signaling pathway is shared among the studied animals. Additionally, galactose metabolism pathway is also found to be commonly involved for proteins derived from camel and horse milk. According to the similarity of casein micelle proteomes, two major sample clusters were classified into ruminant animals (Holstein and Jersey cows, buffaloes, yaks, and goats) and non-ruminants (camels and horses). Our results provide new insights into the protein profile associated with casein micelles and the functionality of the casein micelle from the studied animals. PMID:28240229

In vitro dentine remineralization with a potential salivary phosphoprotein homologue.

PubMed

Romero, Maria Jacinta Rosario H; Nakashima, Syozi; Nikaido, Toru; Sadr, Alireza; Tagami, Junji

2016-08-01

Advantages of introducing a salivary phosphoprotein homologue under standardized in vitro conditions to simulate the mineral-stabilizing properties of saliva have been proposed. This study longitudinally investigates the effects of casein, incorporated as a potential salivary phosphoprotein homologue in artificial saliva (AS) solutions with/without fluoride (F) on in vitro dentine lesion remineralization. Thin sections of bovine root dentine were demineralized and allocated randomly into 6 groups (n=18) having equivalent mineral loss (ΔZ) after transverse microradiography (TMR). The specimens were remineralized using AS solutions containing casein 0μg/ml, F 0ppm (C0-F0); casein 0μg/ml, F 1ppm (C0-F1); casein 10μg/ml, F 0ppm (C10-F0); casein 10μg/ml, F 1ppm (C10-F1); casein 100μg/ml, F 0ppm (C100-F0) or casein 100μg/ml, F 1ppm (C100-F1) for 28days with TMR taken every 7 days. Surface mineral precipitation, evident in group C0-F1, was apparently inhibited in groups with casein incorporation. Repeated measures ANOVA with Bonferroni correction revealed higher ΔZ for non-F and non-casein groups than for their counterparts (p<0.001). Subsequent multiple comparisons showed that mineral gain was higher (p<0.001) with 10μg/ml casein than with 100μg/ml when F was present in the earlier stages of remineralization, with both groups achieving almost complete remineralization after 28 days. Casein is a potential salivary phosphoprotein homologue that could be employed for in vitro dentine remineralization studies. Concentration related effects may be clinically significant and thus must be further examined. Copyright © 2016 Elsevier Ltd. All rights reserved.

Protective role of ascorbic acid in the decontamination of cow milk casein by gamma-irradiation.

PubMed

Kouass Sahbani, Saloua; Klarskov, Klaus; Aloui, Amine; Kouass, Salah; Landoulsi, Ahmed

2013-06-01

The aim of this work was to investigate the protective role of ascorbic acid on irradiation-induced modification of casein. Casein stock solutions were irradiated with increasing doses 2-10 kGy using (60)Co Gamma rays at a dose rate D• = 136.73 Gy/min at room temperature. The total viable microorganism content of cow milk casein was evaluated by Plate Count Agar (PCA) incubation for 48 h at 37°C. Sodium dodecylsulfate gel electrophoresis (SDS-PAGE) and Matrix-Assisted Laser Desorption-Ionization Time-of-Flight mass spectrometry (MALDI-TOF-MS) analysis were used to evaluate the effect of gamma irradiation on casein integrity. Gamma irradiation reduced the bacterial contamination of casein solutions at a lower irradiation dose when performed in the presence of ascorbic acid. The irradiation treatment of casein in the absence of ascorbic acid with a dose of 4 kGy could reduce 99% of the original amount of bacterial colonies. However, in the presence of ascorbic acid the irradiation treatment of casein with a dose lower than 2 kGy could reduce 99% of the original amount of bacterial colonies which suggested that the irradiation dose lower than 2 kGy achieved almost the entire decontamination result. SDS-PAGE and MALDI-TOF-MS analysis showed that ascorbic acid protected cow milk casein from degradation and subsequent aggregation probably by scavenging oxygen and protein radicals produced by the irradiation. It is demonstrated that the combination of gamma irradiation and ascorbic acid produce additive effects, providing acceptable hygienic quality of cow milk casein and protects caseins against Reactive Oxygen Species (ROS) generated, during the irradiation process.

Temperature-dependent dynamics of bovine casein micelles in the range 10-40 °C.

PubMed

Liu, Dylan Z; Weeks, Michael G; Dunstan, David E; Martin, Gregory J O

2013-12-15

Milk is a complex colloidal system that responds to changes in temperature imposed during processing. Whilst much has been learned about the effects of temperature on milk, little is known about the dynamic response of casein micelles to changes in temperature. In this study, a comprehensive physico-chemical study of casein micelles in skim milk was performed between 10 and 40 °C. When fully equilibrated, the amount of soluble casein, soluble calcium and the pH of skim milk all decreased as a function of increasing temperature, whilst the hydration and volume fraction of the casein micelles decreased. The effect of temperature on casein micelle size, as determined by dynamic light scattering and differential centrifugation, was less straightforward. Real-time measurements of turbidity and pH were used to investigate the dynamics of the system during warming and cooling of milk in the range 10-40 °C. Changes in pH are indicative of changes to the mineral system and the turbidity is a measure of alterations to the casein micelles. The pH and turbidity showed that alterations to both the casein micelles and the mineral system occurred very rapidly on warming. However, whilst mineral re-equilibration occurred very rapidly on cooling, changes to the casein micelle structure continued after 40 min of measurement, returning to equilibrium after 16 h equilibration. Casein micelle structure and the mineral system of milk were both dependent on temperature in the range 10-40 °C. The dynamic response of the mineral system to changes in temperature appeared almost instantaneous whereas equilibration of casein was considerably slower, particularly upon cooling. Copyright © 2013 Elsevier Ltd. All rights reserved.

The SNP g.1311T>C associated with the absence of β-casein in goat milk influences CSN2 promoter activity.

PubMed

Cosenza, G; Iannaccone, M; Pico, B A; Ramunno, L; Capparelli, R

2016-10-01

Quantitative individual differences in the amount of β-casein in goat milk are determined by at least nine alleles. In particular, two alleles (CSN2(0) and CSN2(01) ) are associated with an undetectable amount of this protein in milk. The CSN2(01) allele is characterized by a single nucleotide substitution at position 373 of the seventh exon (AJ011018:g.8915C>T), responsible for the formation of a premature stop codon at the 182 position. Herein, we report the contribution of the SNP g.1311T>C, which demonstrates a linkage with the SNP AJ011018:g.8915C>T, to the promoter transcriptional activity. Particularly, we indicate that the nucleotide C at position 1311 negatively affects the promoter activity of the CSN2 gene. © 2016 Stichting International Foundation for Animal Genetics.

Aggregation and structural changes of α(S1)-, β- and κ-caseins induced by homocysteinylation.

PubMed

Stroylova, Yulia Y; Zimny, Jaroslaw; Yousefi, Reza; Chobert, Jean-Marc; Jakubowski, Hieronim; Muronetz, Vladimir I; Haertlé, Thomas

2011-10-01

Elevated homocysteine levels are resulting in N-homocysteinylation of lysyl residues in proteins and they correlate with a number of human pathologies. However, the role of homocysteinylation of lysyl residues is still poorly known. In order to study the features of homocysteinylation of intrinsically unstructured proteins (IUP) bovine caseins were used as a model. α(S1)-, β- and κ-caseins, showing different aggregations and micelle formation, were modified with homocysteine-thiolactone and their physico-chemical properties were studied. Efficiency of homocysteine incorporation was estimated to be about 1.5, 2.1 and 1.3 homocysteyl residues per one β-, α(S1)-, and κ-casein molecule, respectively. Use of intrinsic and extrinsic fluorescent markers such as Trp, thioflavin T and ANS, reveal structural changes of casein structures after homocysteinylation reflected by an increase in beta-sheet content, which in some cases may be characteristic of amyloid-like transformations. CD spectra also show an increase in beta-sheet content of homocysteinylated caseins. Casein homocysteinylation leads in all cases to aggregation. The sizes of aggregates and aggregation rates were dependent on homocysteine thiolactone concentration and temperature. DLS and microscopic studies have revealed the formation of large aggregates of about 1-3μm. Homocysteinylation of α(S1)- and β-caseins results in formation of regular spheres. Homocysteinylated κ-casein forms thin unbranched fibrils about 400-800nm long. In case of κ-casein amyloidogenic effect of homocysteinylation was confirmed by Congo red spectra. Taken together, data indicate that N-homocysteinylation provokes significant changes in properties of native caseins. A comparison of amyloidogenic transformation of 3 different casein types, belonging to the IUP protein family, shows that the efficiency of amyloidogenic transformation upon homocysteinylation depends on micellization capacity, additional disulphide bonds and other structural features. Copyright © 2011 Elsevier B.V. All rights reserved.

Increases in duodenal glutamic acid supply linearly increase small intestinal starch digestion but not nitrogen balance in cattle.

PubMed

Blom, E J; Anderson, D E; Brake, D W

2016-12-01

Small intestinal starch digestion (SISD) in cattle is often limited; however, greater postruminal flow of high-quality protein (e.g., casein) can increase SISD, and Glu can mimic responses to casein for SISD. We evaluated effects of increasing Glu flows to the duodenum on SISD and N retention in cattle. Cattle received (DM basis) continuous duodenal infusion of raw cornstarch (1.5 ± 0.08 kg/d) and 0, 30.9 ± 0.6, 62.4 ± 1.2, or 120.4 ± 3.4 g/d Glu or 387.9 ± 17.5 g/d casein. As expected, the positive control (i.e., casein) increased ( = 0.05) SISD. Interestingly, SISD linearly increased ( = 0.02) with increasing amounts of Glu. Starch flow to the ileum linearly decreased ( = 0.04) in response to greater postruminal Glu and tended to decrease ( = 0.07) with duodenal casein infusion. Ileal flow of ethanol-soluble starch was not affected by duodenal Glu ( = 0.16) or casein ( = 0.42). There was a tendency ( = 0.08) for a quadratic response to Glu for ileal glucose flow with greater flows for intermediate levels of Glu, but casein had no effect ( = 0.81) on glucose flows to the ileum. Greater postruminal supplies of Glu (linear, = 0.05) and casein ( = 0.02) decreased fecal starch flow. Postruminal starch digestion was increased by both casein ( = 0.03) and Glu (linear, = 0.05). Nitrogen intake from feed was not different among treatments ( ≥ 0.23). By design, infusate N increased from 0 to 13 ± 1.5 g/d with greater amounts of Glu, and casein provided 61 ± 1.3 g N/d. Urinary N excretion was not affected ( ≥ 0.30) by postruminal Glu flow, but urine N was increased by casein ( < 0.01). Glutamic acid did not affect N retention ( ≥ 0.34), but casein increased N retention ( < 0.01). However, N retained as a percent of N intake (26.7 ± 1.7%) was not different when cattle were provided Glu ( ≥ 0.16) or casein ( = 0.38).

Role of kappa-casein in the association of denatured whey proteins with casein micelles in heated reconstituted skim milk.

PubMed

Anema, Skelte G

2007-05-02

Reconstituted skim milk at pH from 6.5 to 7.1 was unheated, preheated (68 degrees C/20 min), or heated at 90 degrees C for 20-30 min. On preheating, the size of the casein micelles decreased by about 5-20 nm, with a greater effect at higher pH. The casein micelle size of the heated milk at pH 6.5 increased by about 30 nm when compared to that of the unheated or preheated milk. As the pH was increased before heating, the particle size gradually decreased so that, at pH 7.1, the size was markedly smaller than that for the unheated milk and slightly smaller than that for the preheated milk. High levels (about 85%) of denatured whey protein associated with the casein micelles at pH 6.5, and this level decreased as the pH increased so that, at pH 7.1, low levels (about 15%) were associated with the micelles. Low levels of alphaS-casein and beta-casein were found in the serum regardless of the heat treatment or the pH of the milk. At pH 6.5, low levels (about 10%) of kappa-casein were also found in the milk serum. In the unheated milk, the level of serum kappa-casein increased slightly with increasing pH; in the heated samples, the level of serum kappa-casein increased markedly and linearly with increasing pH so that, at pH 7.1, about 70% of the kappa-casein was in the serum phase. The results of this study indicate that the pH dependence of the levels of serum phase kappa-casein may be responsible for the change in distribution of the whey proteins between the colloidal and serum phases. This is the first report to demonstrate significant levels of dissociation of kappa-casein from the micelles at pH between 6.5 and 6.7, although this dissociation phenomenon is well known on heating milk at high temperatures at pH above 6.7.

The effect of α- or β-casein addition to waxy maize starch on postprandial levels of glucose, insulin, and incretin hormones in pigs as a model for humans

PubMed Central

Kett, Anthony P.; Bruen, Christine M.; O'Halloran, Fiona; Chaurin, Valérie; Lawlor, Peadar G.; O'Mahony, James A.; Giblin, Linda; Fenelon, Mark A.

2012-01-01

Background Starch is a main source of glucose and energy in the human diet. The extent to which it is digested in the gastrointestinal tract plays a major role in variations in postprandial blood glucose levels. Interactions with other biopolymers, such as dairy proteins, during processing can influence both the duration and extent of this postprandial surge. Objective To evaluate the effect of the addition of bovine α- or β-casein to waxy maize starch on changes in postprandial blood glucose, insulin, and incretin hormones [glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1)] in 30 kg pigs used as an animal model for humans. Design Gelatinised starch, starch gelatinised with α-casein, and starch gelatinised with β-casein were orally administered to trained pigs (n = 8) at a level of 60 g of available carbohydrate. Pre- and postprandial glucose measurements were taken every 15 min for the first hour and every 30 min thereafter up to 180 min. Insulin, GIP, and GLP-1 levels were measured in plasma samples up to 90 min postprandial. Results Starch gelatinised with α-casein had a significantly (p < 0.05) lower peak viscosity on pasting and resulted in significantly lower glucose release at 15, 30, and 90 min postprandial compared to starch gelatinised with β-casein. During the first 45-min postprandial, the area under the glucose curve (AUC) for starch gelatinised with α-casein was significantly (p < 0.05) lower than that for starch gelatinised with β-casein. There was also a significant (p < 0.05) difference at T30 in GIP levels in response to the control compared to starch gelatinised with α- or β-casein. Significant (p < 0.05) increases in several free amino acid concentrations were observed on ingestion of either α- or β-casein gelatinised with starch at 30 and 90 min postprandial compared to starch alone. In addition, plasma levels of six individual amino acids were increased on ingestion of starch gelatinised with α-casein compared to ingestion of starch gelatinised with β-casein. Conclusion The presence of casein fractions (α- or β-casein) in gelatinised waxy maize starch affects swelling characteristics, viscosity, and subsequent in vivo digestion as determined by glucose levels in blood postprandial. PMID:22509144

Effects of milk containing only A2 beta casein versus milk containing both A1 and A2 beta casein proteins on gastrointestinal physiology, symptoms of discomfort, and cognitive behavior of people with self-reported intolerance to traditional cows' milk.

PubMed

Jianqin, Sun; Leiming, Xu; Lu, Xia; Yelland, Gregory W; Ni, Jiayi; Clarke, Andrew J

2016-04-02

Cows' milk generally contains two types of β-casein, A1 and A2 types. Digestion of A1 type can yield the peptide β-casomorphin-7, which is implicated in adverse gastrointestinal effects of milk consumption, some of which resemble those in lactose intolerance. This study aimed to compare the effects of milk containing A1 β-casein with those of milk containing only A2 β-casein on inflammation, symptoms of post-dairy digestive discomfort (PD3), and cognitive processing in subjects with self-reported lactose intolerance. Forty-five Han Chinese subjects participated in this double-blind, randomized, 2 × 2 crossover trial and consumed milk containing both β-casein types or milk containing only A2 β-casein. Each treatment period was 14 days with a 14-day washout period at baseline and between treatment periods. Outcomes included PD3, gastrointestinal function (measured by smart pill), Subtle Cognitive Impairment Test (SCIT), serum/fecal laboratory biomarkers, and adverse events. Compared with milk containing only A2 β-casein, the consumption of milk containing both β-casein types was associated with significantly greater PD3 symptoms; higher concentrations of inflammation-related biomarkers and β-casomorphin-7; longer gastrointestinal transit times and lower levels of short-chain fatty acids; and increased response time and error rate on the SCIT. Consumption of milk containing both β-casein types was associated with worsening of PD3 symptoms relative to baseline in lactose tolerant and lactose intolerant subjects. Consumption of milk containing only A2 β-casein did not aggravate PD3 symptoms relative to baseline (i.e., after washout of dairy products) in lactose tolerant and intolerant subjects. Consumption of milk containing A1 β-casein was associated with increased gastrointestinal inflammation, worsening of PD3 symptoms, delayed transit, and decreased cognitive processing speed and accuracy. Because elimination of A1 β-casein attenuated these effects, some symptoms of lactose intolerance may stem from inflammation it triggers, and can be avoided by consuming milk containing only the A2 type of beta casein. ClinicalTrials.gov/NCT02406469.

Pea lectin receptor-like kinase promotes high salinity stress tolerance in bacteria and expresses in response to stress in planta.

PubMed

Joshi, Amita; Dang, Hung Quang; Vaid, Neha; Tuteja, Narendra

2010-01-01

The plant lectin receptor-like kinases (LecRLKs) are involved in various signaling pathways but their role in salinity stress tolerance has not heretofore been well described. Salinity stress negatively affects plant growth/productivity and threatens food security worldwide. Based on functional gene-mining assay, we have isolated 34 salinity tolerant genes out of one million Escherichia coli (SOLR) transformants containing pea cDNAs grown in 0.8 M NaCl. Sequence analysis of one of these revealed homology to LecRLK, which possesses N-myristilation and N-glycosylation sites thus corroborating the protein to be a glycoconjugate. The homology based computational modeling of the kinase domain suggested high degree of conservation with the protein already known to be stress responsive in plants. The NaCl tolerance provided by PsLecRLK to the above bacteria was further confirmed in E. coli (DH5alpha). In planta studies showed that the expression of PsLecRLK cDNA was significantly upregulated in response to NaCl as compared to K(+) and Li(+) ions, suggesting the Na(+) ion specific response. Transcript of the PsLecRLK gene accumulates mainly in roots and shoots. The purified 47 kDa recombinant PsLecRLK-KD (kinase domain) protein has been shown to phosphorylate general substrates like MBP and casein. This study not only suggests the conservation of the cellular response to high salinity stress across prokaryotes and plant kingdom but also provides impetus to develop novel concepts for better understanding of mechanism of stress tolerance in bacteria and plants. It also opens up new avenues for studying practical aspects of plant salinity tolerance for enhanced agricultural productivity.

Heterologous expression of an aspartic protease gene from biocontrol fungus Trichoderma asperellum in Pichia pastoris.

PubMed

Yang, Xiaoxue; Cong, Hua; Song, Jinzhu; Zhang, Junzheng

2013-11-01

Trichoderma asperellum parasitizes a large variety of phytopathogenic fungi. The mycoparasitic activity of T. asperellum depends on the secretion of complex mixtures of hydrolytic enzymes able to degrade the host cell wall and proteases which are a group of enzymes capable of degrading proteins from host. In this study, a full-length cDNA clone of aspartic protease gene, TaAsp, from T. asperellum was obtained and sequenced. The 1,185 bp long cDNA sequence was predicted to encode a 395 amino acid polypeptide with molecular mass of 42.3 kDa. The cDNA of TaAsp was inserted into the pPIC9K vector and transformed into yeast Pichia pastoris GS115 for heterologous expression. A clearly visible band with molecular mass about 42 kDa in the SDS-PAGE gel indicated that the transformant harboring the gene TaAsp had been successfully translated in P. pastoris and produced a recombinant protein. Enzyme characterization test showed that the optimum fermentation time for P. pastoris GS115 transformant was 72 h. Enzyme activity of the recombinant aspartic proteinase remained relatively stable at 25-60 °C and pH 3.0-9.0, which indicated its good prospect of application in biocontrol. The optimal pH value and temperature of the enzyme activity were pH 4.0 and 40 °C, and under this condition, with casein as the substrate, the recombinant protease activity was 18.5 U mL(-1). In order to evaluate antagonistic activity of the recombinant protease against pathogenic fungi, five pathogenic fungi, Fusarium oxysporum, Alternaria alternata, Cytospora chrysosperma, Sclerotinia sclerotiorum and Rhizoctonia solani, were applied to the test of in vitro inhibition of their mycelial growth by culture supernatant of P. pastoris GS115 transformant.

Long-term intake of a high-protein diet increases liver triacylglycerol deposition pathways and hepatic signs of injury in rats.

PubMed

Díaz-Rúa, Rubén; Keijer, Jaap; Palou, Andreu; van Schothorst, Evert M; Oliver, Paula

2017-08-01

Intake of high-protein (HP) diets has increased over the last years, mainly due to their popularity for body weight control. Liver is the main organ handling ingested macronutrients and it is associated with the beginning of different pathologies. We aimed to deepen our knowledge on molecular pathways affected by long-term intake of an HP diet. We performed a transcriptome analysis on liver of rats chronically fed with a casein-rich HP diet and analyzed molecular parameters related to liver injury. Chronic increase in the dietary protein/carbohydrate ratio up-regulated processes related with amino acid uptake/metabolism and lipid synthesis, promoting a molecular environment indicative of hepatic triacylglycerol (TG) deposition. Moreover, changes in expression of genes involved in acid-base maintenance and oxidative stress indicate alterations in the pH balance due to the high acid load of the diet, which has been linked to liver/health damage. Up-regulation of immune-related genes was also observed. In concordance with changes at gene expression level, we observed increased liver TG content and increased serum markers of hepatic injury/inflammation (aspartate transaminase, C-reactive protein and TNF-alpha). Moreover, the HP diet strongly increased hepatic mRNA and protein levels of HSP90, a marker of liver injury. Thus, we show for the first time that long-term consumption of an HP diet, resulting in a high acid load, results in a hepatic transcriptome signature reflecting increased TG deposition and increased signs of health risk (increased inflammation, alterations in the acid-base equilibrium and oxidative stress). Persistence of this altered metabolic status could have unhealthy consequences. Copyright © 2017 Elsevier Inc. All rights reserved.

[Nuclear transfer of goat somatic cells transgenic for human lactoferrin].

PubMed

Li, Lan; Shen, Wei; Pan, Qing-Yu; Min, Ling-Jiang; Sun, Yu-Jiang; Fang, Yong-Wei; Deng, Ji-Xian; Pan, Qing-Jie

2006-12-01

Transgenic animal mammary gland bioreactors are being used to produce recombinant proteins with appropriate post-translational modifications, and nuclear transfer of transgenic somatic cells is a more powerful method to produce mammary gland bioreactor. Here we describe efficient gene transfer and nuclear transfer in goat somatic cells. Gene targeting vector pGBC2LF was constructed by cloning human lactoferrin (LF) gene cDNA into exon 2 of the milk goat beta-casein gene, and the endogenous start condon was replaced by that of human LF gene. Goat fetal fibroblasts were transfected with linearized pGBC2LF and 14 cell lines were positive according to PCR and Southern blot. The transgenic cells were used as donor cells of nuclear transfer, and some of reconstructed embryos could develop to blastocyst in vitro.

A role for cryptochromes in sleep regulation.

PubMed

Wisor, Jonathan P; O'Hara, Bruce F; Terao, Akira; Selby, Chris P; Kilduff, Thomas S; Sancar, Aziz; Edgar, Dale M; Franken, Paul

2002-12-20

The cryptochrome 1 and 2 genes (cry1 and cry2) are necessary for the generation of circadian rhythms, as mice lacking both of these genes (cry1,2-/-) lack circadian rhythms. We studied sleep in cry1,2-/- mice under baseline conditions as well as under conditions of constant darkness and enforced wakefulness to determine whether cryptochromes influence sleep regulatory processes. Under all three conditions, cry1,2-/- mice exhibit the hallmarks of high non-REM sleep (NREMS) drive (i.e., increases in NREMS time, NREMS consolidation, and EEG delta power during NREMS). This unexpected phenotype was associated with elevated brain mRNA levels of period 1 and 2 (per1,2), and albumin d-binding protein (dbp), which are known to be transcriptionally inhibited by CRY1,2. To further examine the relationship between circadian genes and sleep homeostasis, we examined wild type mice and rats following sleep deprivation and found increased levels of per1,2 mRNA and decreased levels of dbp mRNA specifically in the cerebral cortex; these changes subsided with recovery sleep. The expression of per3, cry1,2, clock, npas2, bmal1, and casein-kinase-1epsilon did not change with sleep deprivation. These results indicate that mice lacking cryptochromes are not simply a genetic model of circadian arrhythmicity in rodents and functionally implicate cryptochromes in the homeostatic regulation of sleep.

The poly(A) tail length of casein mRNA in the lactating mammary gland changes depending upon the accumulation and removal of milk.

PubMed Central

Kuraishi, T; Sun, Y; Aoki, F; Imakawa, K; Sakai, S

2000-01-01

The length of casein mRNA from the lactating mouse mammary gland, as assessed on Northern blots, is shorter after weaning, but is elongated following the removal of milk. In order to investigate this phenomenon, the molecular structures of beta- and gamma-casein mRNAs were analysed. The coding and non-coding regions of the two forms were the same length, but the long form of casein mRNA had a longer poly(A) tail than the short form (P<0.05). In order to examine the stability of casein mRNA under identical conditions, casein mRNAs with the long and short poly(A) tails were incubated in the rabbit reticulocyte lysate (RRL) cell-free translation system. Casein mRNA with the long poly(A) tail had a longer half-life than that with the short tail (P<0.05). The beta- and gamma-casein mRNAs were first degraded into 0.92 and 0.81 kb fragments respectively. With undegraded mRNA, the poly(A) tail shortening by exoribonuclease was not observed until the end of the incubation. Northern blot analysis showed that casein mRNA with the long poly(A) tail was protected efficiently from endoribonucleases. We conclude that the length of the poly(A) tail of casein mRNA in the lactating mammary gland changes depending upon the accumulation and removal of the gland's milk, and we show that the longer poly(A) tail potentially protects the mRNA from degradation by endoribonucleases. PMID:10749689

Comparison between pre-exercise casein peptide and intact casein supplementation on glucose tolerance in mice fed a high-fat diet.

PubMed

Matsunaga, Yutaka; Tamura, Yuki; Sakata, Yasuyuki; Nonaka, Yudai; Saito, Noriko; Nakamura, Hirohiko; Shimizu, Takashi; Takeda, Yasuhiro; Terada, Shin; Hatta, Hideo

2018-04-01

We hypothesized that along with exercise, casein peptide supplementation would have a higher impact on improving glucose tolerance than intact casein. Male 6-week-old ICR mice were provided a high-fat diet to induce obesity and glucose intolerance. The mice were randomly divided into 4 treatment groups: control (Con), endurance training (Tr), endurance training with intact casein supplementation (Cas+Tr), and endurance training with casein peptide supplementation (CP+Tr). The mice in each group were orally administrated water, intact casein, or casein peptide (1.0 mg/g body weight, every day), and then subjected to endurance training (15-25 m/min, 60 min, 5 times/week for 4 weeks) on a motor-driven treadmill 30 min after ingestion. Our results revealed that total intra-abdominal fat was significantly lower in CP+Tr than in Con (p < 0.05). Following an oral glucose tolerance test, the blood glucose area under the curve (AUC) was found to be significantly smaller for CP+Tr than for Con (p < 0.05). Moreover, in the soleus muscle, glucose transporter 4 (GLUT4) protein levels were significantly higher in CP+Tr than in Con (p < 0.01). However, intra-abdominal fat, blood glucose AUC, and GLUT4 protein content in the soleus muscle did not alter in Tr and Cas+Tr when compared with Con. These observations suggest that pre-exercise casein peptide supplementation has a higher effect on improving glucose tolerance than intact casein does in mice fed a high-fat diet.

Cardiotrophin-1 Induces Matrix Metalloproteinase-1 in Human Aortic Endothelial Cells

PubMed Central

Tokito, Akinori; Jougasaki, Michihisa; Ichiki, Tomoko; Hamasaki, Shuichi

2013-01-01

Rupture of an atherosclerotic plaque is a key event in the development of cardiovascular disorders, in which matrix metalloproteinase-1 (MMP-1) plays a crucial role by degradation of extracellular matrix resulting in plaque instability. Cardiotrophin-1 (CT-1), a member of interleukin-6-type proinflammatory cytokines, has potent cardiovascular actions and is highly expressed in vascular endothelium, however its role in atherosclerosis has not been fully elucidated to date. The present study was designed to investigate whether CT-1 induces MMP-1 in human aortic endothelial cells (HAECs). Ribonuclease protection assay demonstrated that MMP-1 gene level in HAECs was enhanced by the treatment of CT-1 in a dose- and time-dependent manner. Immunocytochemical staining, Western immunoblot analysis and enzyme-linked immunosorbent assay revealed that CT-1 augmented MMP-1 protein synthesis and secretion. MMP-1 activity assay revealed that MMP-1 present in the supernatant of HAECs was exclusively precursor form. Casein zymography disclosed proteolytic activity in the supernatant of HAECs, which was enhanced by CT-1 treatment. Furthermore, pharmacological inhibitor study indicated the important roles of extracellular signal-regulated kinase (ERK) 1/2, p38 mitogen-activated protein (MAP) kinase, c-Jun N-terminal kinase (JNK) and Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathways in mediating CT-1-induced MMP-1 gene and protein expression. These data reveal for the first time that CT-1 induces the proteolytic potential in HAECs by upregulating MMP-1 expression through ERK1/2, p38 MAP kinase, JNK and JAK/STAT pathways, and suggest that CT-1 may play an important role in the pathophysiology of atherosclerosis and plaque instability. PMID:23935888

The plastid casein kinase 2 phosphorylates Rubisco activase at the Thr-78 site but is not essential for regulation of Rubisco activation state

USDA-ARS?s Scientific Manuscript database

Rubisco activase (RCA) is essential for the activation of Rubisco, the carboxylating enzyme of photosynthesis. In Arabidopsis, RCA is encoded by a single gene (At2g39730) that is alternatively spliced to form a large alpha-RCA and small beta-RCA isoform. The activity of Rubisco is controlled in res...

Casein Kinase 1α Mediates the Degradation of Receptors for Type I and Type II Interferons Caused by Hemagglutinin of Influenza A Virus.

PubMed

Xia, Chuan; Wolf, Jennifer J; Vijayan, Madhuvanthi; Studstill, Caleb J; Ma, Wenjun; Hahm, Bumsuk

2018-04-01

Although influenza A virus (IAV) evades cellular defense systems to effectively propagate in the host, the viral immune-evasive mechanisms are incompletely understood. Our recent data showed that hemagglutinin (HA) of IAV induces degradation of type I IFN receptor 1 (IFNAR1). Here, we demonstrate that IAV HA induces degradation of type II IFN (IFN-γ) receptor 1 (IFNGR1), as well as IFNAR1, via casein kinase 1α (CK1α), resulting in the impairment of cellular responsiveness to both type I and II IFNs. IAV infection or transient HA expression induced degradation of both IFNGR1 and IFNAR1, whereas HA gene-deficient IAV failed to downregulate the receptors. IAV HA caused the phosphorylation and ubiquitination of IFNGR1, leading to the lysosome-dependent degradation of IFNGR1. Influenza viral HA strongly decreased cellular sensitivity to type II IFNs, as it suppressed the activation of STAT1 and the induction of IFN-γ-stimulated genes in response to exogenously supplied recombinant IFN-γ. Importantly, CK1α, but not p38 MAP kinase or protein kinase D2, was proven to be critical for HA-induced degradation of both IFNGR1 and IFNAR1. Pharmacologic inhibition of CK1α or small interfering RNA (siRNA)-based knockdown of CK1α repressed the degradation processes of both IFNGR1 and IFNAR1 triggered by IAV infection. Further, CK1α was shown to be pivotal for proficient replication of IAV. Collectively, the results suggest that IAV HA induces degradation of IFN receptors via CK1α, creating conditions favorable for viral propagation. Therefore, the study uncovers a new immune-evasive pathway of influenza virus. IMPORTANCE Influenza A virus (IAV) remains a grave threat to humans, causing seasonal and pandemic influenza. Upon infection, innate and adaptive immunity, such as the interferon (IFN) response, is induced to protect hosts against IAV infection. However, IAV seems to be equipped with tactics to evade the IFN-mediated antiviral responses, although the detailed mechanisms need to be elucidated. In the present study, we show that IAV HA induces the degradation of the type II IFN receptor IFNGR1 and thereby substantially attenuates cellular responses to IFN-γ. Of note, a cellular kinase, casein kinase 1α (CK1α), is crucial for IAV HA-induced degradation of both IFNGR1 and IFNAR1. Accordingly, CK1α is proven to positively regulate IAV propagation. Thus, this study unveils a novel strategy employed by IAV to evade IFN-mediated antiviral activities. These findings may provide new insights into the interplay between IAV and host immunity to impact influenza virus pathogenicity. Copyright © 2018 American Society for Microbiology.

Caseoperoxidase, Mixed β-Casein-SDS-Hemin-Imidazole Complex: A Nano Artificial Enzyme

PubMed Central

Moosavi-Movahedi, Zainab; Gharibi, Hussein; Hadi-Alijanvand, Hamid; Akbarzadeh, Mohammad; Esmaili, Mansoore; Atri, Maliheh S.; Sefidbakht, Yahya; Bohlooli, Mousa; Nazari, Khodadad; Javadian, Soheila; Hong, Jun; Saboury, Ali A.; Sheibani, Nader; Moosavi-Movahedi, Ali A.

2016-01-01

A novel peroxidase-like artificial enzyme, named “caseoperoxidase”, was biomimetically designed using a nano artificial amino acid apo-protein hydrophobic pocket. This four-component nano artificial enzyme containing heme-imidazole-β-casein-SDS exhibited high activity growth and kcat performance towards the native horseradish peroxidase (HRP) demonstrated by the steady state kinetics using UV-Vis spectrophotometry. The hydrophobicity and secondary structure of the caseoperoxidase were studied by ANS fluorescence and circular dichroism spectroscopy. Camel β-casein (Cβ-casein), with a flexible structure and exalted hydrophobicity, was selected as an appropriate apo-protein for the heme active site using a homology modeling method. Heme docking into the newly obtained Cβ-casein structure indicated one heme was mainly incorporated with Cβ-casein. The presence of a main electrostatic site for the active site in the Cβ-casein was also confirmed by experimental methods through Wyman binding potential and isothermal titration calorimetry. The existence of Cβ-casein protein in this biocatalyst lowered the suicide inactivation, and indicated that the obtained structure has a good protective role for the heme active-site. Additional further experiments confirmed the retention of caseoperoxidase structure and function as an artificial enzyme. PMID:25562503

Caseoperoxidase, mixed β-casein-SDS-hemin-imidazole complex: a nano artificial enzyme.

PubMed

Moosavi-Movahedi, Zainab; Gharibi, Hussein; Hadi-Alijanvand, Hamid; Akbarzadeh, Mohammad; Esmaili, Mansoore; Atri, Maliheh S; Sefidbakht, Yahya; Bohlooli, Mousa; Nazari, Khodadad; Javadian, Soheila; Hong, Jun; Saboury, Ali A; Sheibani, Nader; Moosavi-Movahedi, Ali A

2015-01-01

A novel peroxidase-like artificial enzyme, named "caseoperoxidase", was biomimetically designed using a nano artificial amino acid apo-protein hydrophobic pocket. This four-component nano artificial enzyme containing heme-imidazole-β-casein-SDS exhibited high activity growth and k(cat) performance toward the native horseradish peroxidase demonstrated by the steady state kinetics using UV-vis spectrophotometry. The hydrophobicity and secondary structure of the caseoperoxidase were studied by ANS fluorescence and circular dichroism spectroscopy. Camel β-casein (Cβ-casein) was selected as an appropriate apo-protein for the heme active site because of its innate flexibility and exalted hydrophobicity. This selection was confirmed by homology modeling method. Heme docking into the newly obtained Cβ-casein structure indicated one heme was mainly incorporated with Cβ-casein. The presence of a main electrostatic site for the active site in the Cβ-casein was also confirmed by experimental methods through Wyman binding potential and isothermal titration calorimetry. The existence of Cβ-casein protein in this biocatalyst lowered the suicide inactivation and provided a suitable protective role for the heme active-site. Additional experiments confirmed the retention of caseoperoxidase structure and function as an artificial enzyme.

Amyloid-like fibrils formed from intrinsically disordered caseins: physicochemical and nanomechanical properties.

PubMed

Pan, Kang; Zhong, Qixin

2015-08-07

Amyloid-like fibrils are studied because of their significance in understanding pathogenesis and creating functional materials. Amyloid-like fibrils have been studied by heating globular proteins at acidic conditions. In the present study, intrinsically disordered α-, β-, and κ-caseins were studied to form amyloid-like fibrils at pH 2.0 and 90 °C. No fibrils were observed for α-caseins, and acid hydrolysis was found to be the rate-limiting step of fibrillation of β- and κ-caseins. An increase of β-sheet structure was observed after fibrillation. Nanomechanic analysis of long amyloid-like fibrils using peak-force quantitative nanomechanical atomic force microscopy showed the lowest and highest Young's modulus for β-casein (2.35 ± 0.29 GPa) and κ-casein (4.14 ± 0.66 GPa), respectively. The dispersion with β-casein fibrils had a viscosity more than 10 and 5 times higher than those of κ-casein and β-lactoglobulin, respectively, at 0.1 s(-1) at comparable concentrations. The current findings may assist not only the understanding of amyloid fibril formation but also the development of novel functional materials from disordered proteins.

Aggregation-induced emission of 1,8-naphthalimide-casein micelle: investigation by synchronous spectrographic method.

PubMed

Sun, Yang; Liang, Xuhua; Zhao, Yingyong; Fan, Jun

2013-09-01

A novel 1,8-naphthalimide probe 1, bearing two acetic-acid moieties was synthesized. The acetic-acid groups, docked into the sub-domains of casein micelle and bound with tryptophan residues, and the 1,8-naphthalimide chromophore adsorbed on the surface of casein micelle, forming a supermolecule, 1-casein micelle, which exhibited the aggregation-induced synchronous emission (AISE) characters. The effect of pH on the intensity of supermolecule was investigated, and the result indicated that the emission enhancement was mainly due to the 1,8-naphthalimide chromophore aggregated onto the casein micelle. Based on AISE, a novel casein quantification method was developed, which exhibited a good linear range of 0.05-10.0 μg ml(-1) and 0.07-9.5 μg ml(-1) with the detection limits of 2.8 and 3.0 ng ml(-1) . The effects of metal ions and pH on the system of 1-casein micelle were investigated. The proposed method was applied to determine casein in milk samples, and the results were in good agreement with the result of the Biuret method. Copyright © 2013 Verlag Helvetica Chimica Acta AG, Zürich.

Fractionation and identification of novel antioxidant peptides from buffalo and bovine casein hydrolysates.

PubMed

Shazly, Ahmed Behdal; He, Zhiyong; El-Aziz, Mahmoud Abd; Zeng, Maomao; Zhang, Shuang; Qin, Fang; Chen, Jie

2017-10-01

Buffalo and bovine caseins were hydrolysed by alcalase and trypsin to produce novel antioxidant peptides. The casein hydrolysates were purified using ultrafiltration (UF) and further characterized by RP-HPLC. The fractions produced higher antioxidant activities were identified for their peptides using LC MS/MS. All UF-VI (MW<1kDa) fractions showed higher antioxidant activity. Hydrolysate produced by alcalase for buffalo casein (UF-VI with 54.84-fold purification) showed higher antioxidant activity than that obtained by trypsin. Trypsin hydrolysate contained high amount of hydrophobic amino acids while alcalase hydrolysate consisted mainly of Ser, Arg, Ala and Leu. The antioxidant peptides identified by LC MS/MS were RELEE, MEDNKQ and TVA, EQL in buffalo casein hydrolysates produced by trypsin and alcalase, respectively. Mechanism and reaction pathways of selected antioxidant peptides with ABTS were proposed. Conclusively, buffalo casein provided antioxidant peptides similar to bovine, suggesting that buffalo casein is a novel source of antioxidant. Copyright © 2017 Elsevier Ltd. All rights reserved.

Interactions between tea catechins and casein micelles and their impact on renneting functionality.

PubMed

Haratifar, Sanaz; Corredig, Milena

2014-01-15

Many studies have shown that tea catechins bind to milk proteins. This research focused on the association of tea polyphenols with casein micelles, and the consequences of the interactions on the renneting behaviour of skim milk. It was hypothesized that epigallocatechin-gallate (EGCG), the main catechin present in green tea, forms complexes with the casein micelles and that the association modifies the processing functionality of casein micelles. The binding of EGCG to casein micelles was quantified using HPLC. The formation of catechin-casein micelles complexes affected the rennet induced gelation of milk, and the effect was concentration dependent. Both the primary as well as the secondary stage of gelation were affected. These experiments clearly identify the need for a better understanding of the effect of tea polyphenols on the processing functionality of casein micelles, before milk products can be used as an appropriate platform for delivery of bioactive compounds. Copyright © 2013 Elsevier Ltd. All rights reserved.

Cryo-transmission electron tomography of native casein micelles from bovine milk

PubMed Central

Trejo, R.; Dokland, T.; Jurat-Fuentes, J.; Harte, F.

2013-01-01

Caseins are the principal protein components in milk and an important ingredient in the food industry. In liquid milk, caseins are found as micelles of casein proteins and colloidal calcium nanoclusters. Casein micelles were isolated from raw skim milk by size exclusion chromatography and suspended in milk protein-free serum produced by ultrafiltration (molecular weight cut-off of 3 kDa) of raw skim milk. The micelles were imaged by cryo-electron microscopy and subjected to tomographic reconstruction methods to visualize the 3-dimensional and internal organization of native casein micelles. This provided new insights into the internal architecture of the casein micelle that had not been apparent from prior cryo-transmission electron microscopy studies. This analysis demonstrated the presence of water-filled cavities (~20 to 30 nm in diameter), channels (diameter greater than ~5 nm), and several hundred high-density nanoclusters (6 to 12 nm in diameter) within the interior of the micelles. No spherical protein submicellar structures were observed. PMID:22118067

Influence of succinylation on the conformation of yak casein micelles.

PubMed

Yang, Min; Cui, Na; Fang, Yan; Shi, Ying; Yang, Jitao; Wang, Jiangyu

2015-07-15

Succinylation modifies the physicochemical characteristics and improves the functional properties of proteins. This study assessed the effects of succinylation on the conformation of yak casein micelles with seven degree of modification. The results revealed that succinylation contributed to the dissociation of casein micelles. With the increase of succinylated degree, soluble nitrogen and minerals content increased, while casein micelle size and polydispersity index of micelles decreased. Succinylation affected the spatial conformation of yak casein micelles: turn decreased, ß-sheet and α-helix increased, and irregular structure were non-significantly affected. The intrinsic and ANS fluorescence intensity decreased and the maximum emission wavelength shifted red with increasing succinylation. Based on the results, the structure of yak casein micelles was characteristic of the sub-micelle model. Copyright © 2015 Elsevier Ltd. All rights reserved.

Dietary supplementation with branched-chain amino acids suppresses diethylnitrosamine-induced liver tumorigenesis in obese and diabetic C57BL/KsJ-db/db mice.

PubMed

Iwasa, Junpei; Shimizu, Masahito; Shiraki, Makoto; Shirakami, Yohei; Sakai, Hiroyasu; Terakura, Yoichi; Takai, Koji; Tsurumi, Hisashi; Tanaka, Takuji; Moriwaki, Hisataka

2010-02-01

Obesity and related metabolic abnormalities, including insulin resistance, are risk factors for hepatocellular carcinoma in non-alcoholic steatohepatitis as well as in chronic viral hepatitis. Branched-chain amino acids (BCAA), which improve insulin resistance, inhibited obesity-related colon carcinogenesis in a rodent model, and also reduced the incidence of hepatocellular carcinoma in obese patients with liver cirrhosis. In the present study, we determined the effects of BCAA on the development of diethylnitrosamine (DEN)-induced liver tumorigenesis in obese C57BL/KsJ-db/db (db/db) mice with diabetes mellitus. Male db/db mice were given tap water containing 40 ppm DEN for an initial 2 weeks and thereafter they received a basal diet containing 3.0% of BCAA or casein, which served as a nitrogen content-matched control of BCAA, throughout the experiment. Supplementation with BCAA significantly reduced the total number of foci of cellular alteration, a premalignant lesion of the liver, and the expression of insulin-like growth factor (IGF)-1, IGF-2, and IGF-1 receptor in the liver when compared to the casein supplementation. BCAA supplementation for 34 weeks also significantly inhibited both the development of hepatocellular neoplasms and the proliferation of hepatocytes in comparison to the basal diet or casein-fed groups. Supplementation with BCAA improved liver steatosis and fibrosis and inhibited the expression of alpha-smooth muscle actin in the DEN-treated db/db mice. The serum levels of glucose and leptin decreased by dietary BCAA, whereas the value of the quantitative insulin sensitivity check index increased by this agent, indicating the improvement of insulin resistance and hyperleptinemia. In conclusion, oral BCAA supplementation improves insulin resistance and prevents the development of liver tumorigenesis in obese and diabetic mice.

A high-fat, high-protein diet attenuates the negative impact of casein-induced chronic inflammation on testicular steroidogenesis and sperm parameters in adult mice.

PubMed

Zhao, Jing-Lu; Zhao, Yu-Yun; Zhu, Wei-Jie

2017-10-01

The interaction between obesity and chronic inflammation has been studied. Diet-induced obesity or chronic inflammation could reduce the testicular functions of males. However, the mechanism underlying the reproductive effects of fattening foods in males with or without chronic inflammation still needs further discussion. This study was aimed to investigate the effects of high-fat, high-protein diet on testicular steroidogenesis and sperm parameters in adult mice under physiological and chronic inflammatory conditions. Because casein can trigger a non-infectious systemic inflammatory response, we used casein injection to induce chronic inflammation in male adult Kunming mice. Twenty-four mice were randomly and equally divided into four groups: (i) normal diet+saline (Control); (ii) normal diet+casein (ND+CS); (iii) high-fat, high-protein diet+saline (HFPD+SI); (iv) high-fat, high-protein diet+casein (HFPD+CS). After 8weeks, there was a significant increase in body weight for groups HFPD+SI and HFPD+CS and a decrease in group ND+CS compared with the control. The serum levels of tumor necrosis factor alpha (TNF-α), interleukin-10 (IL-10) and lipid profiles were increased markedly in groups ND+CS, HFPD+SI and HFPD+CS compared with the control. A remarkable reduction of serum adiponectin level occurred in group HFPD+CS compared with group ND+CS. Sperm parameters (sperm count, viability and abnormality) were also adversely affected in groups ND+CS and HFPD+SI. Groups ND+CS and HFPD+SI showed severe pathological changes in testicular tissues. Semiquantitative RT-PCR, Western blot and immunohistochemical staining also showed significant reductions in both testicular mRNA and protein levels of steroidogenic acute regulatory (StAR) and cytochrome P450scc (CYP11A1) in groups HFPD+SI and HFPD+CS compared with the control, whereas testicular mRNA and protein levels of 3β-hydroxysteroid dehydrogenase (3β-HSD) in groups HFPD+SI and HFPD+CS significantly increased. The mRNA and protein levels of the StAR and 3β-HSD in group HFPD+CS were both higher than those of in group ND+CS. These results indicated that Kunming male mice with high-fat, high-protein diet and casein injection for 8weeks can be used to establish a diet-induced obesity and chronic systemic inflammation. The sperm parameters in groups ND+CS and HFPD+SI decreased accompanied by pathological changes of testicular tissue. This resultant effect of reduced serum testosterone levels was associated with the overproduction of TNF-α and IL-10 and down-regulation of StAR and CYP11A1. Under the same casein-induced chronic inflammation condition, the mice with high-fat, high-protein diet had better testicular steroidogenesis activity and sperm parameters compared with the mice in normal diet, indicating that the mice with casein-induced inflammatory injury consuming a high-fat, high-protein diet gained weight normally, reduced serum adiponectin level and increased testosterone production by an upregulation of 3β-HSD expression. High-fat, high-protein diet attenuated the negative impact of casein-induced chronic inflammation on testicular steroidogenesis and sperm parameters. Copyright © 2017 Elsevier Inc. All rights reserved.

Destabilization of Atoh1 by E3 Ubiquitin Ligase Huwe1 and Casein Kinase 1 Is Essential for Normal Sensory Hair Cell Development*

PubMed Central

Cheng, Yen-Fu; Tong, Mingjie; Edge, Albert S. B.

2016-01-01

Proneural basic helix-loop-helix transcription factor, Atoh1, plays a key role in the development of sensory hair cells. We show here that the level of Atoh1 must be accurately controlled by degradation of the protein in addition to the regulation of Atoh1 gene expression to achieve normal cellular patterning during development of the cochlear sensory epithelium. The stability of Atoh1 was regulated by the ubiquitin proteasome system through the action of Huwe1, a HECT-domain, E3 ubiquitin ligase. An interaction between Huwe1 and Atoh1 could be visualized by a proximity ligation assay and was confirmed by co-immunoprecipitation and mass spectrometry. Transfer of a lysine 48-linked polyubiquitin chain to Atoh1 by Huwe1 could be demonstrated both in intact cells and in a cell-free system, and proteasome inhibition or Huwe1 silencing increased Atoh1 levels. The interaction with Huwe1 and polyubiquitylation were blocked by disruption of casein kinase 1 (CK1) activity, and mass spectrometry and mutational analysis identified serine 334 as an important phosphorylation site for Atoh1 ubiquitylation and subsequent degradation. Phosphorylation by CK1 thus targeted the protein for degradation. Development of an extra row of inner hair cells in the cochlea and an approximate doubling in the number of afferent synapses was observed after embryonic or early postnatal deletion of Huwe1 in cochlear-supporting cells, and hair cells died in the early postnatal period when Huwe1 was knocked out in the developing cochlea. These data indicate that the regulation of Atoh1 by the ubiquitin proteasome pathway is necessary for hair cell fate determination and survival. PMID:27542412

pH treatment as an effective tool to select the functional and structural properties of yak milk caseins.

PubMed

Liu, H N; Zhang, C; Zhang, H; Guo, H Y; Wang, P J; Zhu, Y B; Ren, F Z

2013-09-01

Qula is made from yak milk after defatting, acidifying, and drying. Yak milk caseins are purified from Qula by dissolving in alkali solution. The effects of different pH treatments on the functional and structural properties of yak milk caseins were investigated. Over a broad range of pH (from 6.0 to 12.0), functional properties of yak milk caseins, including solubility, emulsifying activities, and thermal characteristics, and the structural properties, including 1-anilino-8-naphthalene-sulfonate fluorescence, turbidity and particle diameter, were evaluated. The results showed that the yak milk casein yield increased as the pH increased from 6.0 to 12.0. The solubility dramatically increased as the pH increased from 6.0 to 8.0, and decreased as the pH increased from 9.0 to 12.0. The changes in emulsifying activity were not significant. Caseins were remarkably heat stable at pH 9.0. The turbidity of the casein solution decreased rapidly as the pH increased from 6.0 to 12.0, and the results suggested that reassembled casein micelles were more compact at low pH than high pH. At pH values higher than 8.0, the yield of yak milk caseins reached more than 80%. The highest solubility was at pH 8.0, the best emulsification was at pH 10.0 and the greatest thermal stability was at pH 9.0. According to the functional characteristics of yak milk caseins, alkali conditions (pH 8.0-10.0) should be selected for optimum production. These results suggested that pH-dependent treatment could be used to modify the properties of yak milk caseins by appropriate selection of the pH level. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Lipopolysaccharide inhibits colonic biotin uptake via interference with membrane expression of its transporter: a role for a casein kinase 2-mediated pathway.

PubMed

Lakhan, Ram; Said, Hamid M

2017-04-01

Biotin (vitamin B7), an essential micronutrient for normal cellular functions, is obtained from both dietary sources as well as gut microbiota. Absorption of biotin in both the small and large intestine is via a carrier-mediated process that involves the sodium-dependent multivitamin transporter (SMVT). Although different physiological and molecular aspects of intestinal biotin uptake have been delineated, nothing is known about the effect of LPS on the process. We addressed this issue using in vitro (human colonic epithelial NCM460 cells) and in vivo (mice) models of LPS exposure. Treating NCM460 cells with LPS was found to lead to a significant inhibition in carrier-mediated biotin uptake. Similarly, administration of LPS to mice led to a significant inhibition in biotin uptake by native colonic tissue. Although no changes in total cellular SMVT protein and mRNA levels were observed, LPS caused a decrease in the fraction of SMVT expressed at the cell surface. A role for casein kinase 2 (CK2) (whose activity was also inhibited by LPS) in mediating the endotoxin effects on biotin uptake and on membrane expression of SMVT was suggested by findings that specific inhibitors of CK2, as well as mutating the putative CK2 phosphorylation site (Thr 78 Ala) in the SMVT protein, led to inhibition in biotin uptake and membrane expression of SMVT. This study shows for the first time that LPS inhibits colonic biotin uptake via decreasing membrane expression of its transporter and that these effects likely involve a CK2-mediated pathway.

Lipopolysaccharide inhibits colonic biotin uptake via interference with membrane expression of its transporter: a role for a casein kinase 2-mediated pathway

PubMed Central

Lakhan, Ram

2017-01-01

Biotin (vitamin B7), an essential micronutrient for normal cellular functions, is obtained from both dietary sources as well as gut microbiota. Absorption of biotin in both the small and large intestine is via a carrier-mediated process that involves the sodium-dependent multivitamin transporter (SMVT). Although different physiological and molecular aspects of intestinal biotin uptake have been delineated, nothing is known about the effect of LPS on the process. We addressed this issue using in vitro (human colonic epithelial NCM460 cells) and in vivo (mice) models of LPS exposure. Treating NCM460 cells with LPS was found to lead to a significant inhibition in carrier-mediated biotin uptake. Similarly, administration of LPS to mice led to a significant inhibition in biotin uptake by native colonic tissue. Although no changes in total cellular SMVT protein and mRNA levels were observed, LPS caused a decrease in the fraction of SMVT expressed at the cell surface. A role for casein kinase 2 (CK2) (whose activity was also inhibited by LPS) in mediating the endotoxin effects on biotin uptake and on membrane expression of SMVT was suggested by findings that specific inhibitors of CK2, as well as mutating the putative CK2 phosphorylation site (Thr78Ala) in the SMVT protein, led to inhibition in biotin uptake and membrane expression of SMVT. This study shows for the first time that LPS inhibits colonic biotin uptake via decreasing membrane expression of its transporter and that these effects likely involve a CK2-mediated pathway. PMID:28052864

Upregulation of heme oxygenase-1 mediates the anti-inflammatory activity of casein glycomacropeptide (GMP) hydrolysates in LPS-stimulated macrophages.

PubMed

Li, Tiange; Cheng, Xue; Du, Min; Chen, Bin; Mao, Xueying

2017-07-19

Recently, we have shown that casein glycomacropeptide hydrolysates (GHP) exhibit both anti-inflammatory and anti-oxidative activities in vitro. However, whether heme oxygenase-1 (HO-1) is involved in the cytoprotective effect of GHP against the inflammatory status remains unclear. Therefore, we hypothesized that HO-1 is a potential target of GHP, which mediates its anti-inflammatory effect. Here, GHP inhibited the intracellular reactive oxygen species (ROS) accumulation and NADPH oxidase 2 (NOX2) expression and enhanced reduced glutathione (GSH) levels in LPS-stimulated RAW264.7 macrophages. GHP also suppressed the expression of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6) and inducible nitric oxide synthase (iNOS) stimulated by lipopolysaccharide (LPS). However, zinc(ii)-protoporphyrin IX (ZnPPIX), a selective inhibitor of HO-1, restored the GHP-mediated suppression of ROS production and NOX2, TNF-α, IL-1β, IL-6 and iNOS expression. GHP treatment inhibited the LPS-induced nuclear transcription factor kappa-B (NF-κB) translocation, which was markedly reversed by ZnPPIX. Furthermore, GHP induced the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), Akt and p38. Pharmacological inhibition of Akt, ERK1/2, and p38 abrogated GHP-induced nuclear localization of NF-E2-related factor-2 (Nrf2) and the expression of HO-1. In summary, GHP inhibits the LPS-induced inflammatory status through upregulating HO-1 expression via PI3K/Akt, ERK1/2 and p38 signaling pathways in RAW264.7 macrophages.

Dose and Time Dependencies in Stress Pathway Responses during Chemical Exposure: Novel Insights from Gene Regulatory Networks.

PubMed

Souza, Terezinha M; Kleinjans, Jos C S; Jennen, Danyel G J

2017-01-01

Perturbation of biological networks is often observed during exposure to xenobiotics, and the identification of disturbed processes, their dynamic traits, and dose-response relationships are some of the current challenges for elucidating the mechanisms determining adverse outcomes. In this scenario, reverse engineering of gene regulatory networks (GRNs) from expression data may provide a system-level snapshot embedded within accurate molecular events. Here, we investigate the composition of GRNs inferred from groups of chemicals with two distinct outcomes, namely carcinogenicity [azathioprine (AZA) and cyclophosphamide (CYC)] and drug-induced liver injury (DILI; diclofenac, nitrofurantoin, and propylthiouracil), and a non-carcinogenic/non-DILI group (aspirin, diazepam, and omeprazole). For this, we analyzed publicly available exposed in vitro human data, taking into account dose and time dependencies. Dose-Time Network Identification (DTNI) was applied to gene sets from exposed primary human hepatocytes using four stress pathways, namely endoplasmic reticulum (ER), NF-κB, NRF2, and TP53. Inferred GRNs suggested case specificity, varying in interactions, starting nodes, and target genes across groups. DILI and carcinogenic compounds were shown to directly affect all pathway-based GRNs, while non-DILI/non-carcinogenic chemicals only affected NF-κB. NF-κB-based GRNs clearly illustrated group-specific disturbances, with the cancer-related casein kinase CSNK2A1 being a target gene only in the carcinogenic group, and opposite regulation of NF-κB subunits being observed in DILI and non-DILI/non-carcinogenic groups. Target genes in NRF2-based GRNs shared by DILI and carcinogenic compounds suggested markers of hepatotoxicity. Finally, we indicate several of these group-specific interactions as potentially novel. In summary, our reversed-engineered GRNs are capable of revealing dose dependent, chemical-specific mechanisms of action in stress-related biological networks.

Small intestinal digestion of raw cornstarch in cattle consuming a soybean hull-based diet is improved by duodenal casein infusion.

PubMed

Brake, D W; Titgemeyer, E C; Bailey, E A; Anderson, D E

2014-09-01

Six duodenally and ileally cannulated steers were used in 3 sequential studies to measure 1) basal nutrient flows from a soybean hull-based diet, 2) small intestinal digestibility of raw cornstarch continuously infused into the duodenum, and 3) responses of small intestinal starch digestion to duodenal infusion of 200 or 400 g/d casein. Our objective was to evaluate responses in small intestinal starch digestion in cattle over time and to measure responses in small intestinal starch digestion to increasing amounts of MP. On average, cattle consumed 3.7 kg/d DM, 68 g/d dietary N, and 70 g/d dietary starch. Starch flow to the duodenum was small (38 g/d), and N flow was 91 g/d. Small intestinal digestibility of duodenal N was 57%, and small intestinal digestion of duodenal starch flow was extensive (92%). Small intestinal starch digestibility was 34% when 1.5 kg/d raw cornstarch was continuously infused into the duodenum. Subsequently, cattle were placed in 1 of 2 replicated Latin squares that were balanced for carryover effects to determine response to casein infusions and time required for adaptation. Duodenal infusion of casein linearly increased (P ≤ 0.05) small intestinal starch digestibility, and small intestinal starch digestion adapted to infusion of casein in 6 d. Ethanol-soluble starch and unpolymerized glucose flowing to the ileum increased linearly (P ≤ 0.05) with increasing infusion of casein. Plasma cholecystokinin was not affected by casein infusion, but circulating levels of glucose were increased by casein supplementation (P ≤ 0.05). Responses in small intestinal starch digestion in cattle adapted to casein within 6 d, and increases in duodenal supply of casein up to 400 g/d increased small intestinal starch digestion in cattle.

Binding of vitamin A by casein micelles in commercial skim milk

PubMed Central

Mohan, M. S.; Jurat-Fuentes, J. L.; Harte, F.

2015-01-01

Recent studies have shown that reassembled micelles formed by caseinates and purified casein fractions (αs- and β-casein) bind to hydrophobic compounds, including curcumin, docosahexaenoic acid, and vitamin D. However, limited research has been done on the binding of hydrophobic compounds by unmodified casein micelles in skim milk. In the present study, we investigated the ability of casein micelles in commercial skim milk to associate with vitamin A (retinyl palmitate), a fat-soluble vitamin commonly used to fortify milk. Milk protein fractions from different commercially available skim milk samples subjected to different processing treatments, including pasteurized, ultrapasteurized, organic pasteurized, and organic ultrapasteurized milks, were separated by fast protein liquid chromatography. The fractions within each peak were combined and freeze-dried. Sodium dodecyl sulfate-PAGE with silver staining was used to identify the proteins present in each of the fractions. The skim milk samples and fractions were extracted for retinyl palmitate and quantified against a standard using normal phase-HPLC. Retinyl palmitate was found to associate with the fraction of skim milk containing caseins, whereas the other proteins (BSA, β-lactoglobulin, α-lactalbumin) did not show any binding. The retinyl palmitate content in the various samples ranged from 1.59 to 2.48 μg of retinyl palmitate per mL of milk. The casein fractions contained between 14 and 40% of total retinyl palmitate in the various milks tested. The variation in the retention of vitamin A by caseins was probably explained by differences in the processing of different milk samples, including thermal treatment, the form of vitamin A emulsion used for fortification, and the point of fortification during processing. Unmodified casein micelles have a strong intrinsic affinity toward the binding of vitamin A used to fortify commercially available skim milks. PMID:23261375

The Effect of Milk Constituents and Crowding Agents on Amyloid Fibril Formation by κ-Casein.

PubMed

Liu, Jihua; Dehle, Francis C; Liu, Yanqin; Bahraminejad, Elmira; Ecroyd, Heath; Thorn, David C; Carver, John A

2016-02-17

When not incorporated into the casein micelle, κ-casein, a major milk protein, rapidly forms amyloid fibrils at physiological pH and temperature. In this study, the effects of milk components (calcium, lactose, lipids, and heparan sulfate) and crowding agents on reduced and carboxymethylated (RCM) κ-casein fibril formation was investigated using far-UV circular dichroism spectroscopy, thioflavin T binding assays, and transmission electron microscopy. Longer-chain phosphatidylcholine lipids, which form the lining of milk ducts and milk fat globules, enhanced RCM κ-casein fibril formation irrespective of whether the lipids were in a monomeric or micellar state, whereas shorter-chain phospholipids and triglycerides had little effect. Heparan sulfate, a component of the milk fat globule membrane and catalyst of amyloid deposition in extracellular tissue, had little effect on the kinetics of RCM κ-casein fibril formation. Major nutritional components such as calcium and lactose also had no significant effect. Macromolecular crowding enhances protein-protein interactions, but in contrast to other fibril-forming species, the extent of RCM κ-casein fibril formation was reduced by the presence of a variety of crowding agents. These data are consistent with a mechanism of κ-casein fibril formation in which the rate-determining step is dissociation from the oligomer to give the highly amyloidogenic monomer. We conclude that the interaction of κ-casein with membrane-associated phospholipids along its secretory pathway may contribute to the development of amyloid deposits in mammary tissue. However, the formation of spherical oligomers such as casein micelles is favored over amyloid fibrils in the crowded environment of milk, within which the occurrence of amyloid fibrils is low.

Effect of calcium concentration on the structure of casein micelles in thin films.

PubMed

Müller-Buschbaum, P; Gebhardt, R; Roth, S V; Metwalli, E; Doster, W

2007-08-01

The structure of thin casein films prepared with spin-coating is investigated as a function of the calcium concentration. Grazing incidence small-angle x-ray scattering and atomic force microscopy are used to probe the micelle structure. For comparison, the corresponding casein solutions are investigated with dynamic light-scattering experiments. In the thin films with added calcium three types of casein structures, aggregates, micelles, and mini-micelles, are observed in coexistence with atomic force microscopy and grazing incidence small-angle x-ray scattering. With increasing calcium concentration, the size of the aggregates strongly increases, while the size of micelles slightly decreases and the size of the mini-micelles increases. This effect is explained in the framework of the particle-stabilizing properties of the hairy layer of kappa-casein surrounding the casein micelles.

Effect of Calcium Concentration on the Structure of Casein Micelles in Thin Films

PubMed Central

Müller-Buschbaum, P.; Gebhardt, R.; Roth, S. V.; Metwalli, E.; Doster, W.

2007-01-01

The structure of thin casein films prepared with spin-coating is investigated as a function of the calcium concentration. Grazing incidence small-angle x-ray scattering and atomic force microscopy are used to probe the micelle structure. For comparison, the corresponding casein solutions are investigated with dynamic light-scattering experiments. In the thin films with added calcium three types of casein structures, aggregates, micelles, and mini-micelles, are observed in coexistence with atomic force microscopy and grazing incidence small-angle x-ray scattering. With increasing calcium concentration, the size of the aggregates strongly increases, while the size of micelles slightly decreases and the size of the mini-micelles increases. This effect is explained in the framework of the particle-stabilizing properties of the hairy layer of κ-casein surrounding the casein micelles. PMID:17496032

Phosphorylation of varicella-zoster virus glycoprotein gpI by mammalian casein kinase II and casein kinase I

DOE Office of Scientific and Technical Information (OSTI.GOV)

Grose, C.; Jackson, W.; Traugh, J.A.

1989-09-01

Varicella-zoster virus (VZV) glycoprotein gpI is the predominant viral glycoprotein within the plasma membranes of infected cells. This viral glycoprotein is phosphorylated on its polypeptide backbone during biosynthesis. In this report, the authors investigated the protein kinases which participate in the phosphorylation events. Under in vivo conditions, VZV gpI was phosphorylated on its serine and threonine residues by protein kinases present within lysates of either VZV-infected or uninfected cells. Because this activity was diminished by heparin, a known inhibitor of casein kinase II, isolated gpI was incubated with purified casein kinase II and shown to be phosphorylated in an inmore » vitro assay containing ({gamma}-{sup 32}P)ATP. The same glycoprotein was phosphorylated when ({sup 32}P)GTP was substituted for ({sup 32}P)ATP in the protein kinase assay. They also tested whether VZV gpI was phosphorylated by two other ubiquitous mammalian protein kinases--casein kinase I and cyclic AMP-dependent kinase--and found that only casein kinase I modified gpI. When the predicted 623-amino-acid sequence of gpI was examined, two phosphorylation sites known to be optimal for casein kinase II were observed. In summary, this study showed that VZV gpI was phosphorylated by each of two mammalian protein kinases (casein kinase I and casein kinase II) and that potential serine-threonine phosphorylation sites for each of these two kinases were present in the viral glycoprotein.« less

Adsorption of Hydrophobin-Protein Mixtures at the Air-Water Interface: The Impact of pH and Electrolyte.

PubMed

Tucker, Ian M; Petkov, Jordan T; Penfold, Jeffrey; Thomas, Robert K; Cox, Andrew R; Hedges, Nick

2015-09-15

The adsorption of the proteins β-casein, β-lactoglobulin, and hydrophobin, and the protein mixtures of β-casein/hydrophobin and β-lactoglobulin/hydrophobin have been studied at the air-water interface by neutron reflectivity, NR. Changing the solution pH from 7 to 2.6 has relatively little impact on the adsorption of hydrophobin or β-lactoglobulin, but results in a substantial change in the structure of the adsorbed layer of β-casein. In β-lactoglobulin/hydrophobin mixtures, the adsorption is dominated by the hydrophobin adsorption, and is independent of the hydrophobin or β-lactoglobulin concentration and solution pH. At pH 2.6, the adsorption of the β-casein/hydrophobin mixtures is dominated by the hydrophobin adsorption over the range of β-casein concentrations studied. At pH 4 and 7, the adsorption of β-casein/hydrophobin mixtures is dominated by the hydrophobin adsorption at low β-casein concentrations. At higher β-casein concentrations, β-casein is adsorbed onto the surface monolayer of hydrophobin, and some interpenetration between the two proteins occurs. These results illustrate the importance of pH on the intermolecular interactions between the two proteins at the interface. This is further confirmed by the impact of PBS, phosphate buffered saline, buffer and CaCl2 on the coadsorption and surface structure. The results provide an important insight into the adsorption properties of protein mixtures and their application in foam and emulsion stabilization.

Inhibition of hydroxyapatite growth by casein, a potential salivary phosphoprotein homologue.

PubMed

Romero, Maria J R H; Nakashima, Syozi; Nikaido, Toru; Ichinose, Shizuko; Sadr, Alireza; Tagami, Junji

2015-08-01

Salivary phosphoproteins are essential in tooth mineral regulation but are often overlooked in vitro. This study aimed to evaluate the effect of casein, as a salivary phosphoprotein homologue, on the deposition and growth of hydroxyapatite (HA) on tooth surfaces. Hydroxyapatite growth was quantified using seeded crystal systems. Artificial saliva (AS) containing HA powder and 0, 10, 20, 50, or 100 μg ml(-1) of casein, or 100 μg ml(-1) of dephosphorylated casein (Dcasein), was incubated for 0-8 h at 37°C, pH 7.2. Calcium concentrations were measured using atomic absorption spectroscopy (AAS). Surface precipitation of HA on bovine enamel and dentine blocks, incubated in similar conditions for 7 d, was examined using field emission scanning electron microscopy (FE-SEM) and transmission electron microscopy (TEM) with selected area electron diffraction (SAED). Casein adsorption was assessed using modified Lowry assays and zeta-potential measurements. The AAS results revealed a concentration-dependent inhibition of calcium consumption. Hydroxyapatite precipitation occurred when no casein was present, whereas precipitation of HA was apparently completely inhibited in casein-containing groups. Adsorption data demonstrated increasingly negative zeta-potential with increased casein concentration and an affinity constant similar to proline-rich proteins with Langmuir modelling. Casein inhibited the deposition and growth of HA primarily through the binding of esterized phosphate to HA active sites, indicating its potential as a mineral-regulating salivary phosphoprotein homologue in vitro. © 2015 Eur J Oral Sci.

The association of low-molecular-weight hydrophobic compounds with native casein micelles in bovine milk

PubMed Central

Cheema, M.; Mohan, M. S.; Campagna, S. R.; Jurat-Fuentes, J. L.; Harte, F. M.

2015-01-01

The agreed biological function of the casein micelles in milk is to carry minerals (calcium, magnesium, and phosphorus) from mother to young along with amino acids for growth and development. Recently, native and modified casein micelles were used as encapsulating and delivery agents for various hydrophobic low-molecular-weight probes. The ability of modified casein micelles to bind certain probes may derive from the binding affinity of native casein micelles. Hence, a study with milk from single cows was conducted to further elucidate the association of hydrophobic molecules into native casein micelles and further understand their biological function. Hydrophobic and hydrophilic extraction followed by ultraperformance liquid chromatography-high resolution mass spectrometry analysis were performed over protein fractions obtained from size exclusion fractionation of raw skim milk. Hydrophobic compounds, including phosphatidylcholine, lyso-phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin, showed strong association exclusively to casein micelles as compared with whey proteins, whereas hydrophilic compounds did not display any preference for their association among milk proteins. Further analysis using liquid chromatography-tandem mass spectrometry detected 42 compounds associated solely with the casein-micelles fraction. Mass fragments in tandem mass spectrometry identified 4 of these compounds as phosphatidylcholine with fatty acid composition of 16:0/18:1, 14:0/16:0, 16:0/16:0, and 18:1/18:0. These results support that transporting low-molecular-weight hydrophobic molecules is also a biological function of the casein micelles in milk. PMID:26074238

Preferential localization of Lactococcus lactis cells entrapped in a caseinate/alginate phase separated system.

PubMed

Léonard, Lucie; Gharsallaoui, Adem; Ouaali, Fahima; Degraeve, Pascal; Waché, Yves; Saurel, Rémi; Oulahal, Nadia

2013-09-01

This study aimed to entrap bioprotective lactic acid bacteria in a sodium caseinate/sodium alginate aqueous two-phase system. Phase diagram at pH=7 showed that sodium alginate and sodium caseinate were not miscible when their concentrations exceeded 1% (w/w) and 6% (w/w), respectively. The stability of the caseinate/alginate two-phase system was also checked at pH values of 6.0 and 5.5. Lactococcus lactis subsp. lactis LAB3 cells were added in a 4% (w/w) caseinate/1.5% (w/w) alginate two-phase system at pH=7. Fluorescence microscopy allowed to observe that the caseinate-rich phase formed droplets dispersed in a continuous alginate-rich phase. The distribution of bacteria in such a system was observed by epifluorescence microscopy: Lc. lactis LAB3 cells stained with Live/Dead(®) Baclight kit™ were located exclusively in the protein phase. Since zeta-potential measurements indicated that alginate, caseinate and bacterial cells all had an overall negative charge at pH 7, the preferential adhesion of LAB cells was assumed to be driven by hydrophobic effect or by depletion phenomena in such biopolymeric systems. Moreover, LAB cells viability was significantly higher in the ternary mixture obtained in the presence of both caseinate and alginate than in single alginate solution. Caseinate/alginate phase separated systems appeared thus well suited for Lc. lactis LAB3 cells entrapment. Copyright © 2013 Elsevier B.V. All rights reserved.

Characterization of a novel ADAM protease expressed by Pneumocystis carinii.

PubMed

Kennedy, Cassie C; Kottom, Theodore J; Limper, Andrew H

2009-08-01

Pneumocystis species are opportunistic fungal pathogens that cause severe pneumonia in immunocompromised hosts. Recent evidence has suggested that unidentified proteases are involved in Pneumocystis life cycle regulation. Proteolytically active ADAM (named for "a disintegrin and metalloprotease") family molecules have been identified in some fungal organisms, such as Aspergillus fumigatus and Schizosaccharomyces pombe, and some have been shown to participate in life cycle regulation. Accordingly, we sought to characterize ADAM-like molecules in the fungal opportunistic pathogen, Pneumocystis carinii (PcADAM). After an in silico search of the P. carinii genomic sequencing project identified a 329-bp partial sequence with homology to known ADAM proteins, the full-length PcADAM sequence was obtained by PCR extension cloning, yielding a final coding sequence of 1,650 bp. Sequence analysis detected the presence of a typical ADAM catalytic active site (HEXXHXXGXXHD). Expression of PcADAM over the Pneumocystis life cycle was analyzed by Northern blot. Southern and contour-clamped homogenous electronic field blot analysis demonstrated its presence in the P. carinii genome. Expression of PcADAM was observed to be increased in Pneumocystis cysts compared to trophic forms. The full-length gene was subsequently cloned and heterologously expressed in Saccharomyces cerevisiae. Purified PcADAMp protein was proteolytically active in casein zymography, requiring divalent zinc. Furthermore, native PcADAMp extracted directly from freshly isolated Pneumocystis organisms also exhibited protease activity. This is the first report of protease activity attributable to a specific, characterized protein in the clinically important opportunistic fungal pathogen Pneumocystis.

Brazilian artisanal ripened cheeses as sources of proteolytic lactic acid bacteria capable of reducing cow milk allergy.

PubMed

Biscola, V; Choiset, Y; Rabesona, H; Chobert, J-M; Haertlé, T; Franco, B D G M

2018-04-13

The objective was to obtain lactic acid bacteria (LAB) capable of hydrolysing immunoreactive proteins in milk, to optimize the hydrolysis, to determine the proteolysis kinetics and to test the safety of the best hydrolytic strain. Brazilian cheese was used as source of LAB capable of hydrolysing main milk allergens. Proteolytic isolates were submitted to RAPD-PCR for the characterization of clonal diversity. Optimized hydrolysis was strain and protein fraction dependent. 16S rDNA sequencing identified three proteolytic strains: Enterococcus faecalis VB43, that hydrolysed α S1 -, α S2 - and β-caseins, α-lactalbumin and β-lactoglobulin (partial hydrolysis), and Pediococcus acidilactici VB90 and Weissella viridescens VB111, that caused partial hydrolysis of α S1 - and α S2 -caseins. Enterococcus faecalis VB43 tested negative for virulence genes asa1, agg, efaA, hyl, esp, cylL L and cylL S but positive for genes ace and gelE. Ethylenediamine tetra-acetic acid inhibited the proteolysis, indicating that the main proteases of E. faecalis VB43 are metalloproteases. Brazilian artisanal cheese is a good source of LAB capable of hydrolysing allergenic proteins in milk. One isolate (E. faecalis VB43) presented outstanding activity against these proteins and lacked most of the tested virulence genes. Enterococcus faecalis VB43 presents good potential for the manufacture of hypoallergenic dairy products. © 2018 The Society for Applied Microbiology.

Purification and molecular characterization of subtilisin-like alkaline protease BPP-A from Bacillus pumilus strain MS-1.

PubMed

Miyaji, T; Otta, Y; Nakagawa, T; Watanabe, T; Niimura, Y; Tomizuka, N

2006-03-01

The present study was conducted by screening zein-degrading bacteria in an attempt to obtain zein-degrading protease. Soil bacteria were screened by formation of a clear zone on zein plates. Characterization of a zein-degrading bacterium indicated a taxonomic affiliation to Bacillus pumilus, and was named MS-1 strain. The strain produced two different types of extracellular proteases, BPP-A and BPP-B. In this study, we purified and characterized BPP-A because it exhibited a higher ability to hydrolyze zein than BPP-B. When casein was used as the substrate, the optimal pH for BPP-A was 11.0. In BPP-A, zein was better substrate than casein at pH 13.0, whereas casein was better one than zein at pH 11.0. The bppA gene encoded a 383-amino acid pre-pro form of BPP-A, and mature BPP-A contained 275 amino acid residues. It was concluded that BPP-A belonged to the subtilisin family. A zein-degrading bacterium assigned to B. pumilus produced two different types of extracellular proteases, BPP-A and BPP-B. BPP-A exhibited an ability to hydrolyze zein in an extreme alkaline condition. This is a first report on screening for zein-degrading micro-organisms. The subtilisin-like protease BPP-A is possible to utilize as an industrial enzyme for the production of zein hydrolysates.

A novel method for separation of caseins from milk by phosphates precipitation.

PubMed

Yen, Chon-Ho; Lin, Yin-Shen; Tu, Ching-Fu

2015-01-01

Milk protein of farm animals is difficult to isolate because of the presence of casein micelles, which are hard to separate from whey by using centrifugation or filtration. Insoluble casein micelles also create an obstacle for purification instruments to operate efficiently. The conventional method, to precipitate caseins by lowering pH to 4.6 and then recover the whey fraction for further purification using chromatography techniques, is not applicable to proteins having an isoelectric point similar to caseins. In addition, the acid condition used for casein removal usually leads to significantly poor yields and reduced biological activities. In this study, a novel method of precipitating caseins under neutral or weak acidic conditions is presented. The method employs a phosphate salt and a freeze-thaw procedure to obtain a casein-free whey protein fraction. This fraction contains more than 90% yield with little loss of bioactivity of the target protein, and is readily available for further chromatographic purification. This method was successfully applied to purify recombinant human factor IX and recombinant hirudin from the milk of transgenic pigs in the presented study. It is an efficient pretreatment approach prior to chromatographic purification of milk protein from farm animals and particularly of great value to collect those recombinants secreted from transgenic livestock.

Nutritional evaluation of caseins and whey proteins and their hydrolysates from Protamex*

PubMed Central

Sindayikengera, Séverin; Xia, Wen-shui

2006-01-01

Whey protein concentrate (WPC 80) and sodium caseinate were hydrolyzed by Protamex to 5%, 10%, 15%, and 20% degree of hydrolysis (DH). WPC 80, sodium caseinate and their hydrolysates were then analyzed, compared and evaluated for their nutritional qualities. Their chemical composition, protein solubility, amino acid composition, essential amino acid index (EAA index), biological value (BV), nutritional index (NI), chemical score, enzymic protein efficiency ratio (E-PER) and in vitro protein digestibility (IVPD) were determined. The results indicated that the enzymatic hydrolysis of WPC 80 and sodium caseinate by Protamex improved the solubility and IVPD of their hydrolysates. WPC 80, sodium caseinate and their hydrolysates were high-quality proteins and had a surplus of essential amino acids compared with the FAO/WHO/UNU (1985) reference standard. The nutritive value of WPC 80 and its hydrolysates was superior to that of sodium caseinate and its hydrolysates as indicated by some nutritional parameters such as the amino acid composition, chemical score, EAA index and predicted BV. However, the E-PER was lower for the WPC hydrolysates as compared to unhydrolyzed WPC 80 but sodium caseinate and its hydrolysates did not differ significantly. The nutritional qualities of WPC 80, sodium caseinate and their hydrolysates were good and make them appropriate for food formulations or as nutritional supplements. PMID:16421963

Alginate/sodium caseinate aqueous-core capsules: a pH-responsive matrix.

PubMed

Ben Messaoud, Ghazi; Sánchez-González, Laura; Jacquot, Adrien; Probst, Laurent; Desobry, Stéphane

2015-02-15

Alginate capsules have several applications. Their functionality depends considerably on their permeability, chemical and mechanical stability. Consequently, the creation of composite system by addition of further components is expected to control mechanical and release properties of alginate capsules. Alginate and alginate-sodium caseinate composite liquid-core capsules were prepared by a simple extrusion. The influence of the preparation pH and sodium caseinate concentration on capsules physico-chemical properties was investigated. Results showed that sodium caseinate influenced significantly capsules properties. As regards to the membrane mechanical stability, composite capsules prepared at pH below the isoelectric point of sodium caseinate exhibited the highest surface Young's modulus, increasing with protein content, explained by potential electrostatic interactions between sodium caseinate amino-groups and alginate carboxylic group. The kinetic of cochineal red A release changed significantly for composite capsules and showed a pH-responsive release. Sodium caseinate-dye mixture studied by absorbance and fluorescence spectroscopy confirmed complex formation at pH 2 by electrostatic interactions between sodium caseinate tryptophan residues and cochineal red sulfonate-groups. Consequently, the release mechanism was explained by membrane adsorption process. This global approach is useful to control release mechanism from macro and micro-capsules by incorporating guest molecules which can interact with the entrapped molecule under specific conditions. Copyright © 2014 Elsevier Inc. All rights reserved.

Casein mediated green synthesis and decoration of reduced graphene oxide

NASA Astrophysics Data System (ADS)

Maddinedi, Sireesh Babu; Mandal, Badal Kumar; Vankayala, Raviraj; Kalluru, Poliraju; Tammina, Sai Kumar; Kiran Kumar, H. A.

This research is mainly focusing on one-step biosynthesis of graphene from graphene oxide and its stabilization using naturally occurring milk protein, casein. The synthesis of casein reduced graphene oxide (CRGO) was completed within 7 h under reflux at 90 °C with the formation of few layered fine graphene nanosheets. UV-Vis, XRD, XPS analysis data revealed the reduction process of the graphene oxide. Results of FT-IR, HPLC and TEM analysis have shown that the ensuing material consists of graphene decorated with casein molecules. Aspartic acid and glutamic acid residue present in casein molecules are responsible for the reduction of graphene oxide.

Effect of casein and inulin addition on physico-chemical characteristics of low fat camel dairy cream.

PubMed

Ziaeifar, Leila; Labbafi Mazrae Shahi, Mohsen; Salami, Maryam; Askari, Gholam R

2018-05-21

The effect of the addition of the camel casein fraction on some physico-chemical properties of low fat camel milk cream was studied. Oil-in-water emulsions, 25, 30, and 35 (w/w) fat, were prepared using inulin, camel skim milk, milk fat and variable percentages of casein (1, 2, and 3% w/w). The droplet size, ζ-potential, surface protein concentration, viscosity and surface tension of low fat dairy creams was measured. Cream containing 2% (w/w) casein had better stability. The modifications in physico-chemical properties appeared to be driven by changes in particle size distribution caused by droplet aggregation. The cream containing 2% casein leads to a gradual decrease in droplet size, as the particle size decreased, apparent viscosity increased. When casein concentration increased, ζ-potential decreased due to combination of c terminal (negative charge) with the surface of fat particles but steric repulsion improved textural properties. Cream with 30% fat and 2% casein had the best result. Copyright © 2018 Elsevier B.V. All rights reserved.

Adsorption of hydrophobin/β-casein mixtures at the solid-liquid interface.

PubMed

Tucker, I M; Petkov, J T; Penfold, J; Thomas, R K; Cox, A R; Hedges, N

2016-09-15

The adsorption behaviour of mixtures of the proteins β-casein and hydrophobin at the hydrophilic solid-liquid surface have been studied by neutron reflectivity. The results of measurements from sequential adsorption and co-adsorption from solution are contrasted. The adsorption properties of protein mixtures are important for a wide range of applications. Because of competing factors the adsorption behaviour of protein mixtures at interfaces is often difficult to predict. This is particularly true for mixtures containing hydrophobin as hydrophobin possesses some unusual surface properties. At β-casein concentrations ⩾0.1wt% β-casein largely displaces a pre-adsorbed layer of hydrophobin at the interface, similar to that observed in hydrophobin-surfactant mixtures. In the composition and concentration range studied here for the co-adsorption of β-casein-hydrophobin mixtures the adsorption is dominated by the β-casein adsorption. The results provide an important insight into how the competitive adsorption in protein mixtures of hydrophobin and β-casein can impact upon the modification of solid surface properties and the potential for a wide range of colloid stabilisation applications. Copyright © 2016 Elsevier Inc. All rights reserved.

Cryo-transmission electron tomography of native casein micelles from bovine milk.

PubMed

Trejo, R; Dokland, T; Jurat-Fuentes, J; Harte, F

2011-12-01

Caseins are the principal protein components in milk and an important ingredient in the food industry. In liquid milk, caseins are found as micelles of casein proteins and colloidal calcium nanoclusters. Casein micelles were isolated from raw skim milk by size exclusion chromatography and suspended in milk protein-free serum produced by ultrafiltration (molecular weight cut-off of 3 kDa) of raw skim milk. The micelles were imaged by cryo-electron microscopy and subjected to tomographic reconstruction methods to visualize the 3-dimensional and internal organization of native casein micelles. This provided new insights into the internal architecture of the casein micelle that had not been apparent from prior cryo-transmission electron microscopy studies. This analysis demonstrated the presence of water-filled cavities (∼20 to 30 nm in diameter), channels (diameter greater than ∼5 nm), and several hundred high-density nanoclusters (6 to 12 nm in diameter) within the interior of the micelles. No spherical protein submicellar structures were observed. Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Effect of experimentally increased protein supply to postpartum dairy cows on plasma protein synthesis, rumen tissue proliferation, and immune homeostasis.

PubMed

Larsen, M; Røntved, C M; Theil, P K; Khatun, M; Lauridsen, C; Kristensen, N B

2017-05-01

The effect of experimentally increasing the postpartum protein supply on plasma protein synthesis, rumen tissue proliferation, and immune homeostasis was studied using 8 periparturient Holstein cows in a complete randomized design. At calving, cows were assigned to abomasal infusion of water (CTRL) or casein (CAS) in addition to a lactation diet. Casein infusion was gradually decreased from 696 ± 1 g/d at +2 d relative to calving (DRTC) to 212 ± 10 g/d at +29 DRTC to avoid excessive supply. Synthesis rate of plasma proteins was measured at -14, +4, +15, and +29 DRTC by measuring [C]Phe isotopic enrichment in arterial plasma free Phe, total plasma proteins, and albumin after 3, 5, and 7 h of jugular ring[C]Phe infusion. Plasma volume was determined at +4 and +29 DRTC by dilution of a [I]BSA dose. Synthesis rate of tissue protein in biopsied rumen papillae was determined by measuring [C]Phe isotopic enrichment, and mRNA expression of selected genes was measured by real-time qPCR. Total and differential leukocyte counts were performed and immune responsiveness of monocytes was evaluated by tumor necrosis factor ɑ (TNFɑ) concentration on ex vivo whole blood stimulation with Escherichia coli lipopolysaccharide (LPS) and responsiveness of T-lymphocytes by interferon γ (IFNγ) concentration on stimulation with Staphylococcus aureus enterotoxin β (SEB). Further, ELISA plasma concentrations of IgM, IgA, and IgG were determined. The DRTC affected the majority of investigated parameters as expected. The CAS treatment increased milk protein yield (P = 0.04), and tended to lower TNFɑ (P = 0.06), and lowered IFNγ (P = 0.03) responsiveness per monocyte and lymphocyte, respectively, compared with CTRL. Further, fractional synthesis rate of albumin was greater at +4 DRTC for CAS compared with CTRL but did not differ by +29 DRTC (interaction: P = 0.01). In rumen papillae, synthesis rate of tissue protein was greater for CAS compared with CTRL (P < 0.01) and mRNA expression of genes for cell proliferation tended to be or were greater for CAS compared with CTRL (P ≤ 0.07). In conclusion, increased postpartum protein supply seem to enhance vital body functions as interpreted from increased liver synthesis of albumin, increased rumen papillae proliferation, and stabilized the ex vivo inflammatory responsiveness of leukocytes. Further studies are needed to enlighten the importance of increased postpartum protein supply in periparturient cows.

Heterocellular interaction enhances recruitment of {alpha} and {beta}-catenins and ZO-2 into functional gap-junction complexes and induces gap junction-dependant differentiation of mammary epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Talhouk, Rabih S.; Mroue, Rana; Mokalled, Mayssa

2008-11-01

Gap junctions (GJ) are required for mammary epithelial differentiation. Using epithelial (SCp2) and myoepithelial-like (SCg6) mouse-derived mammary cells, the role of heterocellular interaction in assembly of GJ complexes and functional differentiation ({beta}-casein expression) was evaluated. Heterocellular interaction is critical for {beta}-casein expression, independent of exogenous basement membrane or cell anchoring substrata. Functional differentiation of SCp2, co-cultured with SCg6, is more sensitive to GJ inhibition relative to homocellular SCp2 cultures differentiated by exogenous basement membrane. Connexin (Cx)32 and Cx43 levels were not regulated across culture conditions; however, GJ functionality was enhanced under differentiation-permissive conditions. Immunoprecipitation studies demonstrated association of junctional complexmore » components ({alpha}-catenin, {beta}-catenin and ZO-2) with Cx32 and Cx43, in differentiation conditions, and additionally with Cx30 in heterocellular cultures. Although {beta}-catenin did not shuttle between cadherin and GJ complexes, increased association between connexins and {beta}-catenin in heterocellular cultures was observed. This was concomitant with reduced nuclear {beta}-catenin, suggesting that differentiation in heterocellular cultures involves sequestration of {beta}-catenin in GJ complexes.« less

Casein kinase 1 (α, δ and ϵ) localize at the spindle poles, but may not be essential for mammalian oocyte meiotic progression

PubMed Central

Qi, Shu-Tao; Wang, Zhen-Bo; Huang, Lin; Liang, Li-Feng; Xian, Ye-Xing; Ouyang, Ying-Chun; Hou, Yi; Sun, Qing-Yuan; Wang, Wei-Hua

2015-01-01

CK1 (casein kinase 1) is a family of serine/threonine protein kinase that is ubiquitously expressed in eukaryotic organism. CK1 members are involved in the regulation of many cellular processes. Particularly, CK1 was reported to phosphorylate Rec8 subunits of cohesin complex and regulate chromosome segregation in meiosis in budding yeast and fission yeast.1-3 Here we investigated the expression, subcellular localization and potential functions of CK1α, CK1δ and CK1ϵ during mouse oocyte meiotic maturation. We found that CK1α, CK1δ and CK1ϵ all concentrated at the spindle poles and co-localized with γ-tubulin in oocytes at both metaphase I (MI) and metaphase II (MII) stages. However, depletion of CK1 by RNAi or overexpression of wild type or kinase-dead CK1 showed no effects on either spindle organization or chromosome segregation during oocyte meiotic maturation. Thus, CK1 is not the kinase that phosphorylates Rec8 cohesin in mammalian oocytes, and CK1 may not be essential for spindle organization and meiotic progression although they localize at spindle poles. PMID:25927854

Soy protein preserves basement membrane integrity through a synergistic effect on nephrin, matrix metalloproteinase and vascular endothelial growth factor.

PubMed

Palanisamy, Nallasamy; Anuradha, Carani Venkataraman

2011-01-01

Soy protein improves renal function and prevents albuminuria in diabetic rats. This study investigates whether the renoprotective effect of soy protein is related to sustenance of basement membrane integrity. Adult male albino rats were randomized into four groups and fed one of the following semi-synthetic diets consisting of corn starch (60%) and casein (20%; CCD), fructose (60%) and casein (20%; FCD), fructose (60%) and soy protein (20%; FSD), or corn starch (60%) and soy protein (20%; CSD). Plasma chemistry and renal changes were analyzed after 60 days. FCD rats displayed metabolic derangements and renal ultrastructural changes. FSD rats showed reduction in type IV collagen, tissue inhibitor for matrix metallo-proteinase-2, vascular endothelial growth factor and tumor necrosis factor-α expression and improved matrix metallo-proteinase expression. Renal architecture was preserved in these rats. Soy protein supplementation not only improved insulin sensitivity but also markedly attenuated renal basement membrane changes in fructose diet-fed rats. These findings provide evidence in support of the use of dietary soy protein in patients with diabetic kidney disease. Copyright © 2011 S. Karger AG, Basel.

Casein micelles and their internal structure.

PubMed

de Kruif, Cornelis G; Huppertz, Thom; Urban, Volker S; Petukhov, Andrei V

2012-01-01

The internal structure of casein micelles was studied by calculating the small-angle neutron and X-ray scattering and static light scattering spectrum (SANS, SAXS, SLS) as a function of the scattering contrast and composition. We predicted experimental SANS, SAXS, SLS spectra self consistently using independently determined parameters for composition size, polydispersity, density and voluminosity. The internal structure of the casein micelles, i.e. how the various components are distributed within the casein micelle, was modeled according to three different models advocated in the literature; i.e. the classical sub-micelle model, the nanocluster model and the dual binding model. In this paper we present the essential features of these models and combine new and old experimental SANS, SAXS, SLS and DLS scattering data with new calculations that predict the spectra. Further evidence on micellar substructure was obtained by internally cross linking the casein micelles using transglutaminase, which led to casein nanogel particles. In contrast to native casein micelles, the nanogel particles were stable in 6M urea and after sequestering the calcium using trisodium citrate. The changed scattering properties were again predicted self consistently. An important result is that the radius of gyration is independent of contrast, indicating that the mass distribution within a casein micelle is homogeneous. Experimental contrast is predicted quite well leading to a match point at a D(2)O volume fraction of 0.41 ratio in SANS. Using SANS and SAXS model calculations it is concluded that only the nanocluster model is capable of accounting for the experimental scattering contrast variation data. All features and trends are predicted self consistently, among which the 'famous' shoulder at a wave vector value Q=0.35 nm(-1) In the nanocluster model, the casein micelle is considered as a (homogeneous) matrix of caseins in which the colloidal calcium phosphate (CCP) nanoclusters are dispersed as very small (about 2 nm) "cherry stones" at an average distance of 18.6 nm. Attached to the surface of the nanoclusters are the centers of phosphorylation (3-5 nearby phosphorylated amino acid residues) of the caseins. The tails of the caseins, much larger than the CCP clusters, then associate to form a protein matrix, which can be viewed as polymer mesh with density fluctuations at the 2 nm scale. The association of the tails is driven by a collection of weak interactions. We explicitly use weak interactions as a collective term for hydrophobic interactions, hydrogen bonding, ion bonding, weak electrostatic Van der Waals attraction and other factors (but not the strong calcium phosphate interaction) leading to self association. The association is highly cooperative and originates in the weak interactions. It is the cooperativety that leads to a stable casein micelle. Invariably, κ-casein is thought to limit the process of self association leading to stabilization of the native casein micelle. Copyright © 2012 Elsevier B.V. All rights reserved.

Casein micelles and their internal structure

DOE Office of Scientific and Technical Information (OSTI.GOV)

De Kruif, Cornelis G; Huppertz, Thom; Urban, Volker S

2012-01-01

The internal structure of casein micelles was studied by calculating the small-angle neutron and X-ray scattering and static light scattering spectrum (SANS, SAXS, SLS) as a function of the scattering contrast and composition. We predicted experimental SANS, SAXS, SLS spectra self consistently using independently determined parameters for composition size, polydispersity, density and voluminosity. The internal structure of the casein micelles, i.e. how the various components are distributed within the casein micelle, was modeled according to three different models advocated in the literature; i.e. the classical sub-micelle model, the nanocluster model and the dual binding model. In this paper we presentmore » the essential features of these models and combine new and old experimental SANS, SAXS, SLS and DLS scattering data with new calculations that predict the spectra. Further evidence on micellar substructure was obtained by internally cross linking the casein micelles using transglutaminase, which led to casein nanogel particles. In contrast to native casein micelles, the nanogel particles were stable in 6 M urea and after sequestering the calcium using trisodium citrate. The changed scattering properties were again predicted self consistently. An important result is that the radius of gyration is independent of contrast, indicating that the mass distribution within a casein micelle is homogeneous. Experimental contrast is predicted quite well leading to a match point at a D{sub 2}O volume fraction of 0.41 ratio in SANS. Using SANS and SAXS model calculations it is concluded that only the nanocluster model is capable of accounting for the experimental scattering contrast variation data. All features and trends are predicted self consistently, among which the 'famous' shoulder at a wave vector value Q = 0.35 nm{sup -1}. In the nanocluster model, the casein micelle is considered as a (homogeneous) matrix of caseins in which the colloidal calcium phosphate (CCP) nanoclusters are dispersed as very small (about 2 nm) 'cherry stones' at an average distance of 18.6 nm. Attached to the surface of the nanoclusters are the centers of phosphorylation (3-5 nearby phosphorylated amino acid residues) of the caseins. The tails of the caseins, much larger than the CCP clusters, then associate to form a protein matrix, which can be viewed as polymer mesh with density fluctuations at the 2 nm scale. The association of the tails is driven by a collection of weak interactions. We explicitly use weak interactions as a collective term for hydrophobic interactions, hydrogen bonding, ion bonding, weak electrostatic Van der Waals attraction and other factors (but not the strong calcium phosphate interaction) leading to self association. The association is highly cooperative and originates in the weak interactions. It is the cooperativety that leads to a stable casein micelle. Invariably, K-casein is thought to limit the process of self association leading to stabilization of the native casein micelle.« less

Influence of succinylation on physicochemical property of yak casein micelles.

PubMed

Yang, Min; Yang, Jitao; Zhang, Yuan; Zhang, Weibing

2016-01-01

Succinylation is a chemical-modification method that affects the physicochemical characteristics and functional properties of proteins. This study assessed the influence of succinylation on the physicochemical properties of yak casein micelles. The results revealed that surface hydrophobicity indices decreased with succinylation. Additionally, denaturation temperature and denaturation enthalpy decreased with increasing succinylation level, except at 82%. The buffering properties of yak casein micelles were affected by succinylation. It was found that chemical modification contributed to a slight shift of the buffering peak towards a lower pH value and a markedly increase of the maximum buffering values of yak casein micelles at pH 4.5-6.0 and pH < 3. Succinylation increased yak casein micellar hydration and whiteness values. The findings obtained from this study will provide the basic information on the physicochemical properties of native and succinylated yak casein micelles. Copyright © 2015 Elsevier Ltd. All rights reserved.

Characteristic aroma components of rennet casein.

PubMed

Karagül-Yüceer, Yonca; Vlahovich, Katrina N; Drake, MaryAnne; Cadwallader, Keith R

2003-11-05

Rennet casein, produced by enzymatic (rennet) precipitation of casein from pasteurized skim milk, is used in both industrial (technical) and food applications. The flavor of rennet casein powder is an important quality parameter; however, the product often contains an odor described as like that of animal/wet dog. Two commercial rennet casein powders were evaluated to determine the compounds responsible for the typical odor. Aroma extracts were prepared by high-vacuum distillation of direct solvent (ether) extracts and analyzed by gas chromatography-olfactometry (GCO), aroma extract dilution analysis (AEDA), and GC-mass spectrometry (MS). Odorants detected by GCO were typical of those previously reported in skim milk powders and consisted mainly of short-chain volatile acids, phenolic compounds, lactones, and furanones. Results of AEDA indicated o-aminoacetophenone to be a potent odorant; however, sensory descriptive sensory analysis of model aroma systems revealed that the typical odor of rennet casein was principally caused by hexanoic acid, indole, guaiacol, and p-cresol.

Effects of ruminal casein and glucose on forage digestion and urea kinetics in beef cattle.

PubMed

Bailey, E A; Titgemeyer, E C; Olson, K C; Brake, D W; Jones, M L; Anderson, D E

2012-10-01

Effects of supplemental glucose and degradable intake protein on nutrient digestion and urea kinetics in steers (Bos taurus) given ad libitum access to prairie hay (4.7% CP) were quantified. Six ruminally and duodenally cannulated steers (initial BW 391 kg) were used in a 4 × 4 Latin square with 2 extra steers. Treatments were arranged as a 2 × 2 factorial and included 0 or 1.2 kg of glucose and 240 or 480 g of casein dosed ruminally once daily. Each period included 9 d for adaptation, 4 d for total fecal and urine collections, and 1 d for ruminal and duodenal sampling. Jugular infusion of (15)N(15)N-urea with measurement of enrichment in urine was used to measure urea kinetics. Glucose reduced forage intake by 18% (P < 0.01), but casein did not affect forage intake (P = 0.69). Glucose depressed (P < 0.01) total tract NDF digestion. Glucose supplementation decreased ruminal pH 2 h after dosing, but the effect was negligible by 6 h (treatment × time; P = 0.01). Providing additional casein increased the ruminal concentration of NH(3), but the increase was less when glucose was supplemented (casein × glucose; P < 0.01). Plasma urea-N was increased (P < 0.01) by additional casein but was reduced (P < 0.01) by glucose. Microbial N flow to the duodenum and retained N increased (P ≤ 0.01) as casein increased, but neither was affected by glucose supplementation. Urea-N entry rate increased (P = 0.03) 50% with increasing casein. Urinary urea-N excretion increased (P < 0.01) as casein increased. The proportion of urea production that was recycled to the gut decreased (P < 0.01) as casein increased. Glucose supplementation decreased (P < 0.01) urinary urea excretion but did not change (P ≥ 0.70) urea production or recycling. The amount of urea-N transferred to the gut and captured by ruminal microbes was less for steers receiving 480 g/d casein with no glucose than for the other 3 treatments (casein × glucose interaction, P = 0.05), which can be attributed to an excess of ruminally available N provided directly to the microbes from the supplement. Overall, the provision of supplemental glucose decreased forage intake and digestibility. Increasing supplemental casein from 240 to 480 g/d increased urea production but decreased the proportion of urea-N recycled to the gut.

The association of low-molecular-weight hydrophobic compounds with native casein micelles in bovine milk.

PubMed

Cheema, M; Mohan, M S; Campagna, S R; Jurat-Fuentes, J L; Harte, F M

2015-08-01

The agreed biological function of the casein micelles in milk is to carry minerals (calcium, magnesium, and phosphorus) from mother to young along with amino acids for growth and development. Recently, native and modified casein micelles were used as encapsulating and delivery agents for various hydrophobic low-molecular-weight probes. The ability of modified casein micelles to bind certain probes may derive from the binding affinity of native casein micelles. Hence, a study with milk from single cows was conducted to further elucidate the association of hydrophobic molecules into native casein micelles and further understand their biological function. Hydrophobic and hydrophilic extraction followed by ultraperformance liquid chromatography-high resolution mass spectrometry analysis were performed over protein fractions obtained from size exclusion fractionation of raw skim milk. Hydrophobic compounds, including phosphatidylcholine, lyso-phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin, showed strong association exclusively to casein micelles as compared with whey proteins, whereas hydrophilic compounds did not display any preference for their association among milk proteins. Further analysis using liquid chromatography-tandem mass spectrometry detected 42 compounds associated solely with the casein-micelles fraction. Mass fragments in tandem mass spectrometry identified 4 of these compounds as phosphatidylcholine with fatty acid composition of 16:0/18:1, 14:0/16:0, 16:0/16:0, and 18:1/18:0. These results support that transporting low-molecular-weight hydrophobic molecules is also a biological function of the casein micelles in milk. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Association between bovine casein antibody and new onset schizophrenia among US military personnel.

PubMed

Niebuhr, David W; Li, Yuanzhang; Cowan, David N; Weber, Natalya S; Fisher, Jared A; Ford, Glen M; Yolken, Robert

2011-05-01

Schizophrenia is a pervasive neuropsychiatric disorder of uncertain etiology. Multiple studies have documented immune activation in individuals with schizophrenia. One antigen capable of inducing a prolonged immune response is bovine casein derived from ingested milk products. Increased levels of casein antibodies have been found in individuals with schizophrenia after diagnosis. This study was directed at determining the potential association between schizophrenia and pre-illness onset levels of immunoglobulin G (IgG) antibodies to bovine casein. Parallel analyses for casein antibody levels with bipolar disorder were included as comparison. Cases were service members who received medical discharges from the military with a schizophrenia diagnosis from 1992 to 2005. Serum specimens were selected for 855 cases and 1165 matched healthy controls. IgG antibodies to bovine whole-casein were measured by solid phase enzyme-linked immunosorbent assays (ELISAs). Hazard ratios (HR) were calculated to examine the associations of casein IgG level with risk of schizophrenia by time to diagnosis and by subjects' initial level. Increasing casein IgG antibody levels among those with a high initial level, drawn before diagnosis, was associated with an 18% increase in the hazard risk of schizophrenia per unit increase (value of low-positive standard) in IgG antibody levels (HR=1.18; 95% CI 1.04, 1.34). This is the first report to identify an association between the risk of schizophrenia and elevated antibodies to bovine casein prior to disease onset. Additional research is required to elucidate the complex genetic environmental interactions involved in the pathogenesis of schizophrenia and to identify potentially modifiable risk factors. Published by Elsevier B.V.

A specific amino acid formula prevents alcoholic liver disease in rodents.

PubMed

Tedesco, Laura; Corsetti, Giovanni; Ruocco, Chiara; Ragni, Maurizio; Rossi, Fabio; Carruba, Michele O; Valerio, Alessandra; Nisoli, Enzo

2018-05-01

Chronic alcohol consumption promotes mitochondrial dysfunction, oxidative stress, defective protein metabolism, and fat accumulation in hepatocytes (liver steatosis). Inadequate amino acid metabolism is worsened by protein malnutrition, frequently present in alcohol-consuming patients, with reduced circulating branched-chain amino acids (BCAAs). Here we asked whether dietary supplementation with a specific amino acid mixture, enriched in BCAAs (BCAAem) and able to promote mitochondrial function in muscle of middle-aged rodents, would prevent mitochondrial dysfunction and liver steatosis in Wistar rats fed on a Lieber-DeCarli ethanol (EtOH)-containing liquid diet. Supplementation of BCAAem, unlike a mixture based on the amino acid profile of casein, abrogated the EtOH-induced fat accumulation, mitochondrial impairment, and oxidative stress in liver. These effects of BCAAem were accompanied by normalization of leucine, arginine, and tryptophan levels, which were reduced in liver of EtOH-consuming rats. Moreover, although the EtOH exposure of HepG2 cells reduced mitochondrial DNA, mitochondrial transcription factors, and respiratory chain proteins, the BCAAem but not casein-derived amino acid supplementation halted this mitochondrial toxicity. Nicotinamide adenine dinucleotide levels and sirtuin 1 (Sirt1) expression, as well as endothelial nitric oxide (eNOS) and mammalian/mechanistic target of rapamycin (mTOR) signaling pathways, were downregulated in the EtOH-exposed HepG2 cells. BCAAem reverted these molecular defects and the mitochondrial dysfunction, suggesting that the mitochondrial integrity obtained with the amino acid supplementation could be mediated through a Sirt1-eNOS-mTOR pathway. Thus a dietary activation of the mitochondrial biogenesis and function by a specific amino acid supplement protects against the EtOH toxicity and preserves the liver integrity in mammals. NEW & NOTEWORTHY Dietary supplementation of a specific amino acid formula prevents both fat accumulation and mitochondrial dysfunction in hepatocytes of alcohol-consuming rats. These effects are accompanied also by increased expression of anti-reactive oxygen species genes. The amino acid-protective effects likely reflect activation of sirtuin 1-endothelial nitric oxide synthase-mammalian target of rapamycin pathway able to regulate the cellular energy balance of hepatocytes exposed to chronic, alcoholic damage.

Antiproliferative activity of tea catechins associated with casein micelles, using HT29 colon cancer cells.

PubMed

Haratifar, S; Meckling, K A; Corredig, M

2014-02-01

Numerous studies have shown that green tea polyphenols display anticancer activities in many organ sites by using different experimental models in rodents and in cultured cell lines in vitro. The present study tested the ability of casein micelles to deliver biologically active concentrations of polyphenols to HT-29 colon cancer cells. Epigallocatechin gallate (EGCG), the major catechin found in green tea, was used as the model molecule, as it has been shown to have antiproliferative activity on colon cancer cells. In the present work, we hypothesized that due to the binding of caseins with EGCG, casein micelles may be an ideal platform for the delivery of this bioactive molecule and that the binding would not affect the bioaccessibility of EGCG. The cytotoxicity and proliferation behavior of HT-29 colon cancer cells when exposed to free EGCG was compared with that of nanoencapsulated EGCG in casein micelles of skim milk. Epigallocatechin gallate-casein complexes were able to decrease the proliferation of HT-29 cancer cells, demonstrating that bioavailability may not be reduced by the nanoencapsulation. As casein micelles may act as protective carriers for EGCG in foods, it was concluded that nanoencapsulation of tea catechins in casein micelles may not diminish their antiproliferative activity on colon cancer cells compared with free tea catechins. Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Evaporative concentration of skimmed milk: effect on casein micelle hydration, composition, and size.

PubMed

Liu, Dylan Z; Dunstan, David E; Martin, Gregory J O

2012-10-01

Understanding the effect of evaporative concentration on casein micelle composition is of high importance for milk processing. Alterations to the hydration, composition and size of casein micelles were investigated in skimmed milk evaporated to concentrations of 12-45% total solids content. The size of casein micelles was determined by dynamic light scattering, and the water content and composition determined by analysis of supernatants and pellets obtained by ultracentrifugation. The mass balance and hydration results showed that during the evaporation process, while micelles were dehydrated, water was removed preferentially from the serum. The amount of soluble casein and calcium in the serum decreased as a function of increasing solids content, indicating a shift of these components to the micelles. The formation of a small proportion of micelle aggregates at high concentrations appeared dependent on the time kept at these concentrations. Upon redilution with water, casein micelles were immediately rehydrated and aggregates were broken up in a matter of minutes. Soluble calcium and pH returned to their original state over a number of hours; however, only a small percentage of original soluble casein returned to the serum over the 5h period investigated. These results showed that casein micelles are significantly affected by evaporative concentration and that the alterations are not completely and rapidly reversible. Copyright © 2012. Published by Elsevier Ltd.

Duodenal supply of glutamate and casein both improve intestinal starch digestion in cattle but by apparently different mechanisms.

PubMed

Brake, D W; Titgemeyer, E C; Anderson, D E

2014-09-01

Greater postruminal flows of protein increase small intestinal starch digestion in cattle. Our objective was to determine if small intestinal starch digestion is increased by duodenal supplementation of AA. We fed 5 duodenally and ileally cannulated steers a low-starch soybean hull-based diet in 5 × 5 Latin square designs and provided continuous duodenal infusion of raw cornstarch in combination with AA or casein and measured small intestinal starch digestion. In Exp. 1 treatments were continuous duodenal infusion of 1) no supplement (control), 2) casein (400 g/d), 3) crystalline AA similar in amount and AA composition to the casein (CASAA), 4) crystalline nonessential AA similar to those provided by casein, or 5) crystalline essential AA similar to those provided by casein. In Exp. 2 treatments were continuous duodenal infusion of 1) no supplement (control), 2) casein (400 g/d), 3) Glu (133 g/d), 4) Phe and Trp plus Met (30.4, 6.5, and 17.5 g/d, respectively; PTM), or 5) a combination of Glu and PTM. Duodenal infusion of casein increased (P ≤ 0.05) small intestinal starch digestion. When CASAA was infused, small intestinal starch digestion was similar (P = 0.30) to casein infusion. Infusion of only nonessential AA tended to increase (P = 0.14) small intestinal starch digestion relative to the control, but infusion of essential AA alone did not affect (P = 0.84) small intestinal starch digestion. In addition, infusion of casein or CASAA increased ileal flows of ethanol-soluble starch (small-chain α-glycosides), but nonessential AA alone were not different than the control. Duodenal infusion of Glu increased (P ≤ 0.05) small intestinal starch digestion, whereas PTM did not. Neither Glu nor PTM increased ileal flow of ethanol-soluble starch, but Glu and PTM provided together tended (P = 0.07) to increase ileal flows of small chain α-glycosides. Our data suggest that Glu alone can increase small intestinal starch digestion in cattle similar to casein, but increases in small intestinal starch digestion in response to Glu are not associated with an increase in ileal flows of small chain α-glycosides.

Identification and subsequent phosphorylation of sequestered partially processed caseins in the lactating guinea-pig mammary gland.

PubMed Central

Boulton, A P; Pascall, J C; Craig, R K

1984-01-01

Golgi and endoplasmic-reticulum fractions were prepared from the lactating guinea-pig mammary gland. The endoplasmic-reticulum fraction was highly active in the processing and sequestration of milk-protein primary translation products. Explants from the lactating gland in organ culture were used to identify milk-protein intermediates present in the secretory pathway, and the timing of the events leading to their post-translational modification. With [35S]methionine, the milk proteins labelled after a short pulse (3 min) were represented by the partially processed (but not phosphorylated) caseins and alpha-lactalbumin sequestered within membrane-bound vesicles. After a 30 min labelling period, higher-Mr caseins with electrophoretic mobilities identical with those of the phosphorylated caseins isolated from milk were identified in the incubation medium, and sequestered within membrane-bound vesicles. Pulse-chase experiments established a precursor-product relationship between these forms. Secretion is apparent approx. 30 min after sequestration. Caseins are highly phosphorylated; removal of the phosphate residues with acid phosphatase results in proteins with increased electrophoretic mobility, similar to those of the partially processed early casein intermediates found sequestered in explants after a 3 min pulse with [35S]methionine, and those sequestered within microsomal membranes after mRNA-directed cell-free protein synthesis. A comparison of the proteins labelled during both short (5 min) and long (30 min) pulses with [35S]methionine and [32P]Pi shows that, in contrast with the 35S-labelled caseins, those labelled with [32P]Pi exhibit only electrophoretic mobilities identical with those of the mature caseins isolated from milk and those identified after long labelling periods with [35S]methionine. No phosphorylated early intermediate forms of caseins were identified. We conclude that the synthesis and post-translational modification of guinea-pig caseins occurs in two stages, (i) an early event involving synthesis and sequestration within the endoplasmic reticulum, an event that involves signal-peptide removal, followed (ii) 10-20 min later by phosphorylation at a different point in the secretory pathway, probably in the Golgi complex. Secretion of the phosphorylated caseins occurs 10-20 min later. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:6477529

Binding of vitamin A by casein micelles in commercial skim milk.

PubMed

Mohan, M S; Jurat-Fuentes, J L; Harte, F

2013-02-01

Recent studies have shown that reassembled micelles formed by caseinates and purified casein fractions (α(s)- and β-casein) bind to hydrophobic compounds, including curcumin, docosahexaenoic acid, and vitamin D. However, limited research has been done on the binding of hydrophobic compounds by unmodified casein micelles in skim milk. In the present study, we investigated the ability of casein micelles in commercial skim milk to associate with vitamin A (retinyl palmitate), a fat-soluble vitamin commonly used to fortify milk. Milk protein fractions from different commercially available skim milk samples subjected to different processing treatments, including pasteurized, ultrapasteurized, organic pasteurized, and organic ultrapasteurized milks, were separated by fast protein liquid chromatography. The fractions within each peak were combined and freeze-dried. Sodium dodecyl sulfate-PAGE with silver staining was used to identify the proteins present in each of the fractions. The skim milk samples and fractions were extracted for retinyl palmitate and quantified against a standard using normal phase-HPLC. Retinyl palmitate was found to associate with the fraction of skim milk containing caseins, whereas the other proteins (BSA, β-lactoglobulin, α-lactalbumin) did not show any binding. The retinyl palmitate content in the various samples ranged from 1.59 to 2.48 µg of retinyl palmitate per mL of milk. The casein fractions contained between 14 and 40% of total retinyl palmitate in the various milks tested. The variation in the retention of vitamin A by caseins was probably explained by differences in the processing of different milk samples, including thermal treatment, the form of vitamin A emulsion used for fortification, and the point of fortification during processing. Unmodified casein micelles have a strong intrinsic affinity toward the binding of vitamin A used to fortify commercially available skim milks. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

A combined spectroscopic, molecular docking and molecular dynamic simulation study on the interaction of quercetin with β-casein nanoparticles.

PubMed

Mehranfar, Fahimeh; Bordbar, Abdol-Khalegh; Parastar, Hadi

2013-10-05

The interaction of quercetin with β-casein nanoparticle micelle was studied at various temperatures in order to do a complete thermodynamic and molecular analysis on the binding process. The results of fluorescence studies showed the possibility of fluorescence energy transfer between excited tryptophan and quercetin. The determined values of critical transfers distance and the mean distance of ligand from Trp-143 residues in β-casein micelle represents a non-radiative energy transfer mechanism for quenching and the existence of a significant interaction between this flavonoid and β-casein nanoparticle. The equilibrium binding of quercetin with β-casein micelle at different temperatures was studied by using UV-Vis absorption spectroscopy. The chemometric analysis (principal component analysis (PCA) and multivariate curve resolution-alternating least squares (MCR-ALS) methods) on spectrophotometric data revealed the existence of two components in solution (quercetin and β-casein-quercetin complex) and resolved their pure concentration and spectral profiles. This information let us to calculate the equilibrium binding constant at various temperatures and the relevant thermodynamic parameters of interaction (enthalpy, entropy and Gibbs free energy) with low uncertainty. The negative values of entropy and enthalpy changes represent the predominate role of hydrogen binding and van der Waals interactions in the binding process. Docking calculations showed the probable binding site of quercetin is located in the hydrophobic core of β-casein where the quercetin molecule is lined by hydrophobic residues and make five hydrogen bonds and several van der Waals contacts with them. Moreover, molecular dynamic (MD) simulation results suggested that this flavonoid can interact with β-casein, without affecting the secondary structure of β-casein. Simulations, molecular docking and experimental data reciprocally supported each other. Copyright © 2013 Elsevier B.V. All rights reserved.

Modification of IgE binding to αS1-casein by proteolytic activity of Enterococcus faecium isolated from Iranian camel milk samples.

PubMed

Kordesedehi, Reihane; Taheri-Kafrani, Asghar; Rabbani-Khorasgani, Mohammad; Kazemi, Rezvan; Mutangadura, Daniel; Haertle, Thomas

2018-06-20

Milk is a perfect source of nutrients for neonates. When breast feeding cannot be done, an infant's alimentation is usually initiated to cow's milk, among the primary foods. It has been reported that about 2.5% of juveniles under the age of 3 years manifest allergic reactions to cow's milk proteins. Among the cow's milk proteins, casein fractions are considered as the strongest allergenic proteins. The proteolytic enzymes of lactic acid bacteria (LAB), during fermentation of dairy products, can break down milk proteins especially caseins and subsequently reduce the immune reactivity of allergenic proteins. In this research, raw bovine and camel milk samples were screened for cocci LAB strains and after isolation, their proteolytic activity against bovine milk caseins were evaluated by SDS-PAGE and RP-HPLC. The potential of cocci LAB strains on α S1 -casein degradation and their potential to break down the principle allergenic epitopes of this protein was detected using indirect competitive ELISA. Molecular identification of the best proteolytic strain was fulfilled by 16S rDNA fragment sequencing with universal primers. The obtained results demonstrated that Enterococcus faecium isolated from raw camel milk samples was the most efficient isolate in hydrolyzing Na-caseinate and α S1 -casein. Hydrolysated α S1 -casein by Enterococcus faecium was also less recognized by IgE of bovine milk allergic patients' sera in comparison with native α S1 -casein. It has been proposed that Enterococcus faecium could be an efficient strain in allergenicity reduction of cow's milk proteins. So it could be an excellent candidate to be potentially used in dairy industries. Copyright © 2018 Elsevier B.V. All rights reserved.

Characterization of casein and poly-L-arginine multilayer films.

PubMed

Szyk-Warszyńska, Lilianna; Kilan, Katarzyna; Socha, Robert P

2014-06-01

Thin films containing casein appear to be a promising material for coatings used in the medical area to promote biomineralization. α- and β-casein and poly-L-arginine multilayer films were formed by the layer-by layer technique and their thickness and mass were analyzed by ellipsometry and quartz crystal microbalance with dissipation monitoring (QCM-D). (PLArg/casein) films deposited in 0.15M NaCl exhibit fast (exponential-like) growth of the film thickness with the number of layers. The resulting films were c.a. 10 times thicker than obtained for poly-L-arginine and natural polyanions. We investigated the effect of the type of casein used for the film formation, finding that films with α-casein were slightly thicker than ones with β-casein. The effect of polyethylene imine anchoring layer on the thickness and mass of adsorbed films was similar as for linear polyelectrolyte pairs. Thickness of "wet" films was c.a. two times larger than measured after drying that suggests their large hydration. The analysis of the mass of films during their post-treatment with the solutions of various ionic strength and pH provided the information concerning films stability. Films remain stable in the neutral and weakly basic conditions that includes HEPES buffer, which is widely used in cell culture and biomedical experiments. At the conditions of high ionic strength films swell but their swelling is reversible. Films containing caseins as polyanion appear to be more elastic and the same time more viscous than one formed with polyelectrolyte pairs. XPS elemental analysis confirmed binding of calcium ions by the casein embedded in the multilayers. Copyright © 2014 Elsevier Inc. All rights reserved.

The binding of orally dosed hydrophobic active pharmaceutical ingredients to casein micelles in milk.

PubMed

Cheema, M; Hristov, A N; Harte, F M

2017-11-01

Casein proteins (α S1 -, α S2 -, β- and κ-casein) account for 80% of the total protein content in bovine milk and form casein micelles (average diameter = 130 nm, approximately 10 15 micelles/mL). The affinity of native casein micelles with the 3 hydrophobic active pharmaceutical ingredients (API), meloxicam [351.4 g/mol; log P = 3.43; acid dissociation constant (pK a ) = 4.08], flunixin (296.2 g/mol; log P = 4.1; pK a = 5.82), and thiabendazole (201.2 g/mol; log P = 2.92; pK a = 4.64), was evaluated in bovine milk collected from dosed Holstein cows. Native casein micelles were separated from raw bovine milk by mild techniques such as ultracentrifugation, diafiltration, isoelectric point precipitation (pH 4.6), and size exclusion chromatography. Acetonitrile extraction of hydrophobic API was then done, followed by quantification using HPLC-UV. For the API or metabolites meloxicam, 5-hyroxy flunixin and 5-hydroxy thiabendazole, 31 ± 3.90, 31 ± 1.3, and 28 ± 0.5% of the content in milk was associated with casein micelles, respectively. Less than ∼5.0% of the recovered hydrophobic API were found in the milk fat fraction, and the remaining ∼65% were associated with the whey/serum fraction. A separate in vitro study showed that 66 ± 6.4% of meloxicam, 29 ± 0.58% of flunixin, 34 ± 0.21% of the metabolite 5-hyroxy flunixin, 50 ± 4.5% of thiabendazole, and 33 ± 3.8% of metabolite 5-hydroxy thiabendazole was found partitioned into casein micelles. Our study supports the hypothesis that casein micelles are native carriers for hydrophobic compounds in bovine milk. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

/sup 54/Mn absorption and excretion in rats fed soy protein and casein diets

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, D.Y.; Johnson, P.E.

1989-02-01

Rats were fed diets containing either soy protein or casein and different levels of manganese, methionine, phytic acid, or arginine for 7 days and then fed test meals labeled with 2 microCi of 54Mn after an overnight fast. Retention of 54Mn in each rat was measured every other day for 21 days using a whole-body counter. Liver manganese was higher (P less than 0.0001) in soy protein-fed rats (8.8 micrograms/g) than in casein-fed rats (5.2 micrograms/g); manganese superoxide dismutase activity also was higher in soy protein-fed rats than in casein-fed rats (P less than 0.01). There was a significant interactionmore » between manganese and protein which affected manganese absorption and biologic half-life of 54Mn. In a second experiment, rats fed soy protein-test meals retained more 54Mn (P less than 0.001) than casein-fed rats. Liver manganese (8.3 micrograms/g) in the soy protein group was also higher than that (5.7 micrograms/g) in the casein group (P less than 0.0001), but manganese superoxide dismutase activity was unaffected by protein. Supplementation with methionine increased 54Mn retention from both soy and casein diets (P less than 0.06); activity of manganese superoxide dismutase increased (P less than 0.05) but liver manganese did not change. The addition of arginine to casein diets had little effect on manganese bioavailability. Phytic acid affected neither manganese absorption nor biologic half-life in two experiments, but it depressed liver manganese in one experiment. These results suggest that neither arginine nor phytic acid was the component in soy protein which made manganese more available from soy protein diets than casein diets.« less

RAMA casein zymography: Time-saving and highly sensitive casein zymography for MMP7 and trypsin.

PubMed

Yasumitsu, Hidetaro; Ozeki, Yasuhiro; Kanaly, Robert A

2016-11-01

To detect metalloproteinase-7 (MMP7), zymography is conducted using a casein substrate and conventional CBB stain. It has disadvantages because it is time consuming and has low sensitivity. Previously, a sensitive method to detect MMP7 up to 30 pg was reported, however it required special substrates and complicated handlings. RAMA casein zymography described herein is rapid, sensitive, and reproducible. By applying high-sensitivity staining with low substrate conditions, the staining process is completed within 1 h and sensitivity was increased 100-fold. The method can detect 10 pg MMP7 by using commercially available casein without complicated handlings. Moreover, it increases detection sensitivity for trypsin. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

AFM imaging of milk casein micelles: evidence for structural rearrangement upon acidification.

PubMed

Ouanezar, Mustapha; Guyomarc'h, Fanny; Bouchoux, Antoine

2012-03-20

Milk casein micelles are natural association colloids that we all encounter in everyday life, yet we still lack an accurate description of their internal structure and the interactions that stabilize it. In this letter, we provide for the first time detailed images of intact casein micelles as obtained through atomic force microscopy under liquid conditions close to physiological. The micelles appear as heterogeneous raspberry-like particles, which is consistent with a hierarchical/spongelike structure made of connected 10-40 nm dense casein regions. Upon in situ acidification to pH 5, the micelles decrease in size and lose their surface heterogeneities, indicating that this structure is highly sensitive to variations in mineral content and caseins net charge.

Dysregulation of Protease and Protease Inhibitors in a Mouse Model of Human Pelvic Organ Prolapse

PubMed Central

Budatha, Madhusudhan; Silva, Simone; Montoya, Teodoro Ignacio; Suzuki, Ayako; Shah-Simpson, Sheena; Wieslander, Cecilia Karin; Yanagisawa, Masashi; Word, Ruth Ann; Yanagisawa, Hiromi

2013-01-01

Mice deficient for the fibulin-5 gene (Fbln5−/−) develop pelvic organ prolapse (POP) due to compromised elastic fibers and upregulation of matrix metalloprotease (MMP)-9. Here, we used casein zymography, inhibitor profiling, affinity pull-down, and mass spectrometry to discover additional protease upregulated in the vaginal wall of Fbln5−/− mice, herein named V1 (25 kDa). V1 was a serine protease with trypsin-like activity similar to protease, serine (PRSS) 3, a major extrapancreatic trypsinogen, was optimum at pH 8.0, and predominantly detected in estrogenized vaginal epithelium of Fbln5−/− mice. PRSS3 was (a) localized in epithelial secretions, (b) detected in media of vaginal organ culture from both Fbln5−/− and wild type mice, and (c) cleaved fibulin-5 in vitro. Expression of two serine protease inhibitors [Serpina1a (α1-antitrypsin) and Elafin] was dysregulated in Fbln5−/− epithelium. Finally, we confirmed that PRSS3 was expressed in human vaginal epithelium and that SERPINA1 and Elafin were downregulated in vaginal tissues from women with POP. These data collectively suggest that the balance between proteases and their inhibitors contributes to support of the pelvic organs in humans and mice. PMID:23437119

Protein quality and the protein to carbohydrate ratio within a high fat diet influences energy balance and the gut microbiota in C57BL/6J mice.

PubMed

McAllan, Liam; Skuse, Peter; Cotter, Paul D; O'Connor, Paula; Cryan, John F; Ross, R Paul; Fitzgerald, Gerald; Roche, Helen M; Nilaweera, Kanishka N

2014-01-01

Macronutrient quality and composition are important determinants of energy balance and the gut microbiota. Here, we investigated how changes to protein quality (casein versus whey protein isolate; WPI) and the protein to carbohydrate (P/C) ratio within a high fat diet (HFD) impacts on these parameters. Mice were fed a low fat diet (10% kJ) or a high fat diet (HFD; 45% kJ) for 21 weeks with either casein (20% kJ, HFD) or WPI at 20%, 30% or 40% kJ. In comparison to casein, WPI at a similar energy content normalised energy intake, increased lean mass and caused a trend towards a reduction in fat mass (P = 0.08), but the protein challenge did not alter oxygen consumption or locomotor activity. WPI reduced HFD-induced plasma leptin and liver triacylglycerol, and partially attenuated the reduction in adipose FASN mRNA in HFD-fed mice. High throughput sequence-based analysis of faecal microbial populations revealed microbiota in the HFD-20% WPI group clustering closely with HFD controls, although WPI specifically increased Lactobacillaceae/Lactobacillus and decreased Clostridiaceae/Clostridium in HFD-fed mice. There was no effect of increasing the P/C ratio on energy intake, but the highest ratio reduced HFD-induced weight gain, fat mass and plasma triacylglycerol, non-esterified fatty acids, glucose and leptin levels, while it increased lean mass and oxygen consumption. Similar effects were observed on adipose mRNA expression, where the highest ratio reduced HFD-associated expression of UCP-2, TNFα and CD68 and increased the diet-associated expression of β3-AR, LPL, IR, IRS-1 and GLUT4. The P/C ratio also impacted on gut microbiota, with populations in the 30/40% WPI groups clustering together and away from the 20% WPI group. Taken together, our data show that increasing the P/C ratio has a dramatic effect on energy balance and the composition of gut microbiota, which is distinct from that caused by changes to protein quality.

Protein Quality and the Protein to Carbohydrate Ratio within a High Fat Diet Influences Energy Balance and the Gut Microbiota In C57BL/6J Mice

PubMed Central

McAllan, Liam; Skuse, Peter; Cotter, Paul D.; Connor, Paula O'; Cryan, John F.; Ross, R. Paul; Fitzgerald, Gerald; Roche, Helen M.; Nilaweera, Kanishka N.

2014-01-01

Macronutrient quality and composition are important determinants of energy balance and the gut microbiota. Here, we investigated how changes to protein quality (casein versus whey protein isolate; WPI) and the protein to carbohydrate (P/C) ratio within a high fat diet (HFD) impacts on these parameters. Mice were fed a low fat diet (10% kJ) or a high fat diet (HFD; 45% kJ) for 21 weeks with either casein (20% kJ, HFD) or WPI at 20%, 30% or 40% kJ. In comparison to casein, WPI at a similar energy content normalised energy intake, increased lean mass and caused a trend towards a reduction in fat mass (P = 0.08), but the protein challenge did not alter oxygen consumption or locomotor activity. WPI reduced HFD-induced plasma leptin and liver triacylglycerol, and partially attenuated the reduction in adipose FASN mRNA in HFD-fed mice. High throughput sequence-based analysis of faecal microbial populations revealed microbiota in the HFD-20% WPI group clustering closely with HFD controls, although WPI specifically increased Lactobacillaceae/Lactobacillus and decreased Clostridiaceae/Clostridium in HFD-fed mice. There was no effect of increasing the P/C ratio on energy intake, but the highest ratio reduced HFD-induced weight gain, fat mass and plasma triacylglycerol, non-esterified fatty acids, glucose and leptin levels, while it increased lean mass and oxygen consumption. Similar effects were observed on adipose mRNA expression, where the highest ratio reduced HFD-associated expression of UCP-2, TNFα and CD68 and increased the diet-associated expression of β3-AR, LPL, IR, IRS-1 and GLUT4. The P/C ratio also impacted on gut microbiota, with populations in the 30/40% WPI groups clustering together and away from the 20% WPI group. Taken together, our data show that increasing the P/C ratio has a dramatic effect on energy balance and the composition of gut microbiota, which is distinct from that caused by changes to protein quality. PMID:24520424

The pressure-induced, lactose-dependent changes in the composition and size of casein micelles.

PubMed

Wang, Pengjie; Jin, Shaoming; Guo, Huiyuan; Zhao, Liang; Ren, Fazheng

2015-04-15

The effects of lactose on the changes in the composition and size of casein micelles induced by high-pressure treatment and the related mechanism of action were investigated. Dispersions of ultracentrifuged casein micelle pellets with 0-10% (w/v) lactose were subjected to high pressure (400 MPa) at 20 °C for 40 min. The results indicated that the level of non-sedimentable caseins was positively related to the amount of lactose added prior to pressure treatment, and negatively correlated to the size. A mechanism for the pressure-induced, lactose-dependent changes in the casein micelles is proposed. Lactose inhibits the hydrophobic interactions between the micellar fragments during or after pressure release, through the hydrophilic layer formed by their hydrogen bonds around the micellar fragments. In addition, lactose does not favour the association between calcium and the casein aggregates after pressure release. Due to these two functions, lactose inhibited the formation of larger micelles after pressure treatment. Copyright © 2014 Elsevier Ltd. All rights reserved.

Acid gelation properties of heated skim milk as a result of enzymatically induced changes in the micelle/serum distribution of the whey protein/kappa-casein aggregates.

PubMed

Guyomarc'h, Fanny; Renan, Marie; Chatriot, Marc; Gamerre, Valérie; Famelart, Marie-Hélène

2007-12-26

Changes in the acid gelation properties of skim milk as a result of variations in the micelle/serum distribution of the heat-induced whey protein/kappa-casein aggregates, induced by the combination of heat treatment and limited renneting, were investigated. No dramatic change in the zeta potential or the isoelectric point of the casein micelles was suggested, whether the aggregates were all attached to the casein micelle or not. Fluorescence intensity measurement using 8-anilino-1-naphthalenesulfonic acid (ANS) showed that the heat-induced aggregates were highly hydrophobic. Dynamic oscillation viscosimetry showed that acid gelation using glucono-delta-lactone (GDL) started at a higher pH value in prerenneted milk. However, no change in the gelation profile of skim milk could be related to the proportion of aggregates bound to the surface of the casein micelles. The results support the idea of an early interaction between the serum aggregates and the casein micelles on acidification.

Mesoscale crystallization of calcium phosphate nanostructures in protein (casein) micelles.

PubMed

Thachepan, Surachai; Li, Mei; Mann, Stephen

2010-11-01

Aqueous micelles of the multi-protein calcium phosphate complex, casein, were treated at 60°C and pH 7 over several months. Although partial dissociation of the micelles into 12 nm sized amorphous calcium phosphate (ACP)/protein nanoparticles occurred within a period of 14 days, crystallization of the ACP nanoclusters into bundles of hydroxyapatite (HAP) nanofilaments was not observed until after 12 weeks. The HAP nanofilaments were formed specifically within the partially disrupted protein micelles suggesting a micelle-mediated pathway of mesoscale crystallization. Similar experiments using ACP-containing synthetic micelles prepared from ß-casein protein alone indicated that co-aligned bundles of HAP nanofilaments were produced within the protein micelle interior after 24 hours at temperatures as low as 35°C. The presence of Mg²(+) ions in the casein micelles, as well as a possible synergistic effect associated with the multi-protein nature of the native aggregates, could account for the marked inhibition in mesoscale crystallization observed in the casein micelles compared with the single-component b-casein constructs.

Rennet-induced gelation of concentrated milk in the presence of sodium caseinate: differences between milk concentration using ultrafiltration and osmotic stressing.

PubMed

Krishnankutty Nair, P; Corredig, M

2015-01-01

Concentrating milk is a common unit operation in the dairy industry. With the reduction of water, the particles interact more frequently with each other and the functionality of the casein micelles may depend on the interactions occurring during concentration. The objective of this research was to investigate the effect of concentration on the renneting properties of the casein micelles by comparing 2 concentration methods: ultrafiltration and osmotic stressing. Both methods selectively concentrate the protein fraction of milk, while the composition of the soluble phase is unaltered. To evaluate possible differences in the rearrangements of the casein micelles during concentration, renneting properties were evaluated with or without the addition of soluble caseins, added either before or after concentration. The results indicate that casein micelles undergo rearrangements during concentration and that shear during membrane filtration may play a role in affecting the final properties of the milk. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Effect of the coexistence of sodium caseinate and Tween 20 as stabilizers of food emulsions at acidic pH.

PubMed

Perugini, Luisa; Cinelli, Giuseppe; Cofelice, Martina; Ceglie, Andrea; Lopez, Francesco; Cuomo, Francesca

2018-02-05

In the present investigation the properties of edible nanoemulsions were studied. Sodium caseinate represents a good candidate for food emulsion preparations thanks to its surface-active properties and because it is perceived as a natural product by consumers. Nevertheless, it is very sensitive to acidic pH close to its isoelectric point and, if used as emulsion stabilizer, this aspect can negatively affect the emulsion stability. In order to prevent this drawback, sodium caseinate was used in combination with a non-ionic surfactant (Tween 20) as emulsifier of oil/water nanoemulsions. For these reasons, nanoemulsions stabilized by Tween 20, sodium caseinate and by a blend of the two emulsifiers were studied and compared according to their response to pH variations. Nanoemulsions were characterized for size of the dispersed phase with variation of time and temperature, for their rheological properties, for surface charge as a function of pH and for protein fluorescence. Noticeably, it was ascertained that, at pH close to caseinate isoelectric point, emulsions stabilized with the blend of caseinate and Tween 20 were more stable, compared with emulsions stabilized only with sodium caseinate. Such behavior was explained according to the composition of the emulsifiers at the oil/water interface where, at acidic pH, the presence of Tween 20 ensured the steric stabilization thus improving the role of sodium caseinate as emulsion stabilizer. Copyright © 2018 Elsevier B.V. All rights reserved.

Structural and shear characteristics of adsorbed sodium caseinate and monoglyceride mixed monolayers at the air-water interface.

PubMed

Rodríguez Patino, Juan M; Cejudo Fernández, Marta; Carrera Sánchez, Cecilio; Rodríguez Niño, Ma Rosario

2007-09-01

The structural and shear characteristics of mixed monolayers formed by an adsorbed Na-caseinate film and a spread monoglyceride (monopalmitin or monoolein) on the previously adsorbed protein film have been analyzed. Measurements of the surface pressure (pi)-area (A) isotherm and surface shear viscosity (eta(s)) were obtained at 20 degrees C and at pH 7 in a modified Wilhelmy-type film balance. The structural and shear characteristics of the mixed films depend on the surface pressure and on the composition of the mixed film. At surface pressures lower than the equilibrium surface pressure of Na-caseinate (at pipi(e)(CS) have important repercussions on the shear characteristics of the mixed films.

Dietary phenylalanine-improved intestinal barrier health in young grass carp (Ctenopharyngodon idella) is associated with increased immune status and regulated gene expression of cytokines, tight junction proteins, antioxidant enzymes and related signalling molecules.

PubMed

Feng, Lin; Li, Wen; Liu, Yang; Jiang, Wei-Dan; Kuang, Sheng-Yao; Jiang, Jun; Tang, Ling; Wu, Pei; Tang, Wu-Neng; Zhang, Yong-An; Zhou, Xiao-Qiu

2015-08-01

The present work evaluated the effects of dietary phenylalanine (Phe) on the intestinal immune response, tight junction proteins transcript abundance, and the gene expression of immune- and antioxidant-related signalling molecules in the intestine. In addition, the dietary Phe (and Phe + Tyr) requirement of young grass carp (Ctenopharyngodon idella) was also estimated. Fish were fed fish meal-casein-gelatin based diets (302.3 g crude protein kg(-1)) containing 3.4 (basal diet), 6.1, 9.1, 11.5, 14.0 and 16.8 g Phe kg(-1) with a fixed amount of 10.7 g tyrosine kg(-1) for 8 weeks. The results showed that Phe deficiency or excess Phe reduced the lysozyme and acid phosphatase activities and complement C 3 content in the intestine (P < 0.05). Moreover, zonula occludens-1 (ZO-1), occludin and claudin c mRNA levels were highest in the fish fed the diet containing 11.5 g Phe kg(-1) (P < 0.05). However, claudin 12 and claudin b mRNA levels were not significantly affected by dietary Phe (P > 0.05). Gene expression of interleukin-10 (IL-10), transforming growth factor-β1 (TGF-β1), target of rapamycin (TOR) and inhibitor of nuclear factor κBα (IκBα) in proximal intestine (PI), mid intestine (MI) and distal intestine (DI) increased as dietary Phe increased up to 6.1, 9.1, 11.5 and 14.0 g kg(-1), respectively (P < 0.05). However, interleukin-8 (IL-8), tumour necrosis factor-α (TNF-α) and nuclear factor-κB p65 (NF-κB p65) mRNA levels showed opposite tendencies. In addition, the mRNA level of superoxide dismutase (SOD) was significantly lower in the intestinal tissue of the group fed a diet with Phe levels of 16.8 g kg(-1) than in those of other groups (P < 0.05). The expression of NF-E2-related factor 2 (Nrf2) gene was increased as dietary Phe increased up to 9.1 g kg(-1) (P < 0.05). In conclusion, Phe improved intestinal immune status, and regulated gene expression of cytokines, tight junction proteins, antioxidant enzymes, NF-κB p65, IκBα, TOR, and Nrf2 in the fish intestine. Based on the quadratic regression analysis of lysozyme activity at a 95% maximum, the dietary Phe requirement of young grass carp (256-629 g) was estimated to be 8.31 g kg(-1), corresponding to 2.75 g 100 g(-1) protein. Copyright © 2015 Elsevier Ltd. All rights reserved.

Molecular characterization of a gene POLR2H encoded an essential subunit for RNA polymerase II from the Giant Panda (Ailuropoda Melanoleuca).

PubMed

Du, Yu-Jie; Hou, Yi-Ling; Hou, Wan-Ru

2013-02-01

The Giant Panda is an endangered and valuable gene pool in genetic, its important functional gene POLR2H encodes an essential shared peptide H of RNA polymerases. The genomic DNA and cDNA sequences were cloned successfully for the first time from the Giant Panda (Ailuropoda melanoleuca) adopting touchdown-PCR and reverse transcription polymerase chain reaction (RT-PCR), respectively. The length of the genomic sequence of the Giant Panda is 3,285 bp, including five exons and four introns. The cDNA fragment cloned is 509 bp in length, containing an open reading frame of 453 bp encoding 150 amino acids. Alignment analysis indicated that both the cDNA and its deduced amino acid sequence were highly conserved. Protein structure prediction showed that there was one protein kinase C phosphorylation site, four casein kinase II phosphorylation sites and one amidation site in the POLR2H protein, further shaping advanced protein structure. The cDNA cloned was expressed in Escherichia coli, which indicated that POLR2H fusion with the N-terminally His-tagged form brought about the accumulation of an expected 20.5 kDa polypeptide in line with the predicted protein. On the basis of what has already been achieved in this study, further deep-in research will be conducted, which has great value in theory and practical significance.

Estimation of genetic parameters and detection of chromosomal regions affecting the major milk proteins and their post translational modifications in Danish Holstein and Danish Jersey cattle.

PubMed

Buitenhuis, Bart; Poulsen, Nina A; Gebreyesus, Grum; Larsen, Lotte B

2016-08-02

In the Western world bovine milk products are an important protein source in human diet. The major proteins in bovine milk are the four caseins (CN), αS1-, αS2-, β-, and k-CN and the two whey proteins, β-LG and α-LA. It has been shown that both the amount of specific CN and their isoforms including post-translational modifications (PTM) influence technological properties of milk. Therefore, the aim of this study was to 1) estimate genetic parameters for individual proteins in Danish Holstein (DH) (n = 371) and Danish Jersey (DJ) (n = 321) milk, and 2) detect genomic regions associated with specific milk protein and their different PTM forms using a genome-wide association study (GWAS) approach. For DH, high heritability estimates were found for protein percentage (0.47), casein percentage (0.43), k-CN (0.77), β-LG (0.58), and α-LA (0.40). For DJ, high heritability estimates were found for protein percentage (0.70), casein percentage (0.52), and α-LA (0.44). The heritability for G-k-CN, U-k-CN and GD was higher in the DH compared to the DJ, whereas the heritability for the PD of αS1-CN was lower in DH compared to DJ, whereas the PD for αS2-CN was higher in DH compared to DJ. The GWAS results for the main milk proteins were in line what has been earlier published. However, we showed that there were SNPs specifically regulating G-k-CN in DH. Some of these SNPs were assigned to casein protein kinase genes (CSNK1G3, PRKCQ). The genetic analysis of the major milk proteins and their PTM forms revealed that these were heritable in both DH and DJ. In DH, genomic regions specific for glycosylation of k-CN were detected. Furthermore, genomic regions for the major milk proteins confirmed the regions on BTA6 (casein cluster), BTA11 (PEAP), and BTA14 (DGAT1) as important regions influencing protein composition in milk. The results from this study provide confidence that it is possible to breed for specific milk protein including the different PTM forms.

Classification of Lactococcus lactis cell envelope proteinase based on gene sequencing, peptides formed after hydrolysis of milk, and computer modeling.

PubMed

Børsting, M W; Qvist, K B; Brockmann, E; Vindeløv, J; Pedersen, T L; Vogensen, F K; Ardö, Y

2015-01-01

Lactococcus lactis strains depend on a proteolytic system for growth in milk to release essential AA from casein. The cleavage specificities of the cell envelope proteinase (CEP) can vary between strains and environments and whether the enzyme is released or bound to the cell wall. Thirty-eight Lc. lactis strains were grouped according to their CEP AA sequences and according to identified peptides after hydrolysis of milk. Finally, AA positions in the substrate binding region were suggested by the use of a new CEP template based on Streptococcus C5a CEP. Aligning the CEP AA sequences of 38 strains of Lc. lactis showed that 21 strains, which were previously classified as group d, could be subdivided into 3 groups. Independently, similar subgroupings were found based on comparison of the Lc. lactis CEP AA sequences and based on normalized quantity of identified peptides released from αS1-casein and β-casein. A model structure of Lc. lactis CEP based on the crystal structure of Streptococcus C5a CEP was used to investigate the AA positions in the substrate-binding region. New AA positions were suggested, which could be relevant for the cleavage specificity of CEP; however, these could only explain 2 out of 3 found subgroups. The third subgroup could be explained by 1 to 5 AA positions located opposite the substrate binding region. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

21 CFR 520.1157 - Iodinated casein tablets.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Iodinated casein tablets. 520.1157 Section 520.1157 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS ORAL DOSAGE FORM NEW ANIMAL DRUGS § 520.1157 Iodinated casein...

Alpha-lactalbumin and casein-glycomacropeptide do not affect iron absorption from formula in healthy term infants

USDA-ARS?s Scientific Manuscript database

Iron absorption from infant formula is relatively low. Alpha-lactalbumin and casein-glycomacropeptide have been suggested to enhance mineral absorption. We therefore assessed the effect of alpha-lactalbumin and casein-glycomacropeptide on iron absorption from infant formula in healthy term infants. ...

Crystal structure of casein kinase-1, a phosphate-directed protein kinase.

PubMed Central

Xu, R M; Carmel, G; Sweet, R M; Kuret, J; Cheng, X

1995-01-01

The structure of a truncated variant of casein kinase-1 from Schizosaccharomyces pombe, has been determined in complex with MgATP at 2.0 A resolution. The model resembles the 'closed', ATP-bound conformations of the cyclin-dependent kinase 2 and the cAMP-dependent protein kinase, with clear differences in the structure of surface loops that impart unique features to casein kinase-1. The structure is of unphosphorylated, active conformation of casein kinase-1 and the peptide-binding site is fully accessible to substrate. Images PMID:7889932

Characterization of casein and poly-l-arginine multilayer films

NASA Astrophysics Data System (ADS)

Szyk-Warszyńska, Lilianna; Kilan, Katarzyna; Socha, Robert P.

2014-06-01

Thin films containing casein appear to be a promising material for coatings used in the medical area to promote biomineralization. alfa- and beta-casein and poly-L-arginine multilayer films were formed by the layer-by layer technique and their thickness and mass were analyzed by ellipsometry and quartz crystal microbalance with dissipation monitoring (QCM-D). We investigated the effect of the type of casein used for the film formation and of the polyethyleneimine anchoring layer on the thickness and mass of adsorbed films. The analysis of the mass of films during their post-treatment with the solutions of various ionic strength and pH provided the information concerning films stability, while the XPS elemental analysis confirmed binding of calcium ions by the casein embedded in the multilayers.

Structural characterization of casein micelles: shape changes during film formation.

PubMed

Gebhardt, R; Vendrely, C; Kulozik, U

2011-11-09

The objective of this study was to determine the effect of size-fractionation by centrifugation on the film structure of casein micelles. Fractionated casein micelles in solution were asymmetrically distributed with a small distribution width as measured by dynamic light scattering. Films prepared from the size-fractionated samples showed a smooth surface in optical microscopy images and a homogeneous microstructure in atomic force micrographs. The nano- and microstructure of casein films was probed by micro-beam grazing incidence small angle x-ray scattering (μGISAXS). Compared to the solution measurements, the sizes determined in the film were larger and broadly distributed. The measured GISAXS patterns clearly deviate from those simulated for a sphere and suggest a deformation of the casein micelles in the film. © 2011 IOP Publishing Ltd

Evaluation of Structure, Chaperone-Like Activity and Allergenicity of Reduced Glycated Adduct of Bovine β-casein.

PubMed

Yousefi, Reza; Ferdowsi, Leila; Tavaf, Zohreh; Sadeghian, Tanaz; Tamaddon, Ali M; Moghtaderi, Mozhgan; Pourpak, Zahra

2017-01-01

Milk has a potent reducing environment with an important quantity of sugar levels. In the current study β-casein was glycated in the presence of D-glucose and sodium cyanoborohydride as a reducing agent. Then, the reduced glucitol adduct of β-casein was used for the structural and functional analyses using different spectroscopic techniques. The results of fluorescence and far ultraviolet circular dichroism assessments suggest important structural alteration upon non-enzymatic glycation of β-casein. In addition, the chaperone activity, micellization properties and antioxidant activity of this protein were altered upon glucose modification. Also, as a result of reduced glycation, the allergenicity profile of this protein remained largely unchanged. Additional to its energetic and nutritional values, β-casein has important functional properties. The native structure of this protein is important to perform accurately its biological functions. Non-enzymatic glycation under reducing state was capable to alter both structural and functional aspects of β-casein. Due to effective reducing environment and significant quantity of reducing sugar of human milk, similar structural and functional alterations are most likely to occur upon reducing glycation of β-casein in vivo. Also, these changes might be even intensified during chronic hyperglycemia in diabetic mothers. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

The influence of casein haplotype on morphometric characteristics of fat globules and fatty acid composition of milk in Italian Holstein cows.

PubMed

Perna, Annamaria; Intaglietta, Immacolata; Simonetti, Amalia; Gambacorta, Emilio

2016-04-01

The aim of this work was to investigate the effect of casein haplotypes (αS1-, β-, and κ-caseins) on morphometric characteristics of fat globules and fatty acid composition of Italian Holstein milk. Casein haplotypes were determined by isoelectric focusing; milk fat globule size was measured by using a fluorescence microscope; and fatty acid profile was determined by gas chromatography. Casein haplotype significantly affected the fat globule size, the percentage incidence of each globule size class on total measured milk fat globules, and fatty acid composition. A higher incidence of smaller milk fat globules was associated with the BB-A(2)A(2)-BB genotype (αS1-, β-, and κ-casein haplotypes, respectively), whereas small globules were not detected in BB-A(2)A(1)-AA milk, but that milk had the highest percentage of large globules. A higher content of monounsaturated fatty acids was associated with the BB-A(2)A(2)-AB genotype, whereas higher contents of conjugated linoleic acid and docosahexaenoic acid were detected in BB-A(1)A(1)-AA milk. Our results indicate that casein haplotype could affect fat characteristics and, therefore, the nutritional and technological quality of milk. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Mesoscale crystallization of calcium phosphate nanostructures in protein (casein) micelles

NASA Astrophysics Data System (ADS)

Thachepan, Surachai; Li, Mei; Mann, Stephen

2010-11-01

Aqueous micelles of the multi-protein calcium phosphate complex, casein, were treated at 60 °C and pH 7 over several months. Although partial dissociation of the micelles into 12 nm sized amorphous calcium phosphate (ACP)/protein nanoparticles occurred within a period of 14 days, crystallization of the ACP nanoclusters into bundles of hydroxyapatite (HAP) nanofilaments was not observed until after 12 weeks. The HAP nanofilaments were formed specifically within the partially disrupted protein micelles suggesting a micelle-mediated pathway of mesoscale crystallization. Similar experiments using ACP-containing synthetic micelles prepared from β-casein protein alone indicated that co-aligned bundles of HAP nanofilaments were produced within the protein micelle interior after 24 hours at temperatures as low as 35 °C. The presence of Mg2+ ions in the casein micelles, as well as a possible synergistic effect associated with the multi-protein nature of the native aggregates, could account for the marked inhibition in mesoscale crystallization observed in the casein micelles compared with the single-component β-casein constructs.Aqueous micelles of the multi-protein calcium phosphate complex, casein, were treated at 60 °C and pH 7 over several months. Although partial dissociation of the micelles into 12 nm sized amorphous calcium phosphate (ACP)/protein nanoparticles occurred within a period of 14 days, crystallization of the ACP nanoclusters into bundles of hydroxyapatite (HAP) nanofilaments was not observed until after 12 weeks. The HAP nanofilaments were formed specifically within the partially disrupted protein micelles suggesting a micelle-mediated pathway of mesoscale crystallization. Similar experiments using ACP-containing synthetic micelles prepared from β-casein protein alone indicated that co-aligned bundles of HAP nanofilaments were produced within the protein micelle interior after 24 hours at temperatures as low as 35 °C. The presence of Mg2+ ions in the casein micelles, as well as a possible synergistic effect associated with the multi-protein nature of the native aggregates, could account for the marked inhibition in mesoscale crystallization observed in the casein micelles compared with the single-component β-casein constructs. Electronic supplementary information (ESI) available: Particle size histograms, TEM, EDX and electron diffraction data. See DOI: 10.1039/c0nr00158a

Extracellular Matrix Signaling from the Cellular Membrane Skeleton to the Nuclear Skeleton: A Model of Gene Regulation

PubMed Central

Lelièvre, Sophie; Weaver, Valerie M.; Bissell, Mina J.

2010-01-01

It is well established that cells must interact with their microenvironment and that such interaction is crucial for coordinated function and homeostasis. However, how cells receive and integrate external signals leading to gene regulation is far from understood. It is now appreciated that two classes of cooperative signals are implicated: a soluble class including hormones and growth factors and a class of insoluble signals emanating from the extracellular matrix (ECM) directly through contact with the cell surface. Using 3-dimensional culture systems and transgenic mice, we have been able to identify some of the elements of this ECM-signaling pathway responsible for gene regulation in rodent mammary gland differentiation and involution. Our major observations are 1) the requirement for a laminin-rich basement membrane; 2) the existence of a cooperative signaling pathway between basement membrane and the lactogenic hormone prolactin (PRL); 3) the importance of β1-integrins and bHLH transcription factor(s) and the presence of DNA response elements (exemplified by BCE-1, located on a milk protein gene, β-casein); and 4) the induction of mammary epithelial cell programmed cell death following degradation of basement membrane. We hypothesize that this cooperative signaling between ECM and PRL may be achieved through integrin- and laminin-directed restructuring of the cytoskeleton leading to profound changes in nuclear architecture and transcription factor localization. We postulate that the latter changes allow the prolactin signal to activate transcription of the β-casein gene. To further understand the molecular mechanisms underlying ECM and hormonal cooperative signaling, we are currently investigating ECM regulation of a “solid-state” signaling pathway including ECM fiber proteins, plasma membrane receptors, cytoskeleton, nuclear matrix and chromatin. We further postulate that disruption of such a pathway may be implicated in cell disorders including transformation and carcinogenesis. PMID:8701089

THE ANTIGENIC PROPERTIES OF SPLIT PRODUCTS OF CASEIN

PubMed Central

Gay, Frederick P.; Robertson, T. Brailsford

1912-01-01

We draw the following conclusions from our experiments on the antigenic properties of chemically pure casein and some of its split products. Casein and paranuclein have distinct antigenic properties, particularly as shown by their ability to sensitize guinea pigs for subsequent anaphylactic intoxication by each other or by milk. This sensitizing ability and a corresponding ability to intoxicate are indistinguishably equivalent, under the conditions employed. On immunizing rabbits by repeated injections of paranuclein or of casein, and subsequently testing their sera for precipitins and fixation antibodies, it was found that casein apparently produces them much more readily, giving an antiserum that reacted (fixation) in very high dilution with casein (0.000,000,1 of a 1 per cent. solution), but much less strongly with paranuclein. Only one of two antiparanuclein sera showed the presence of antibodies to paranuclein by the delicate fixation reaction, and that in relatively small amounts. The two antibodies to casein and to paranuclein are, in the case of casein quantitatively, and in the case of paranuclein absolutely specific. A solution of the products of complete peptic digestion of casein fails to sensitize to paranuclein and gives no fixation reaction with an anticasein or antiparanuclein serum. It intoxicates animals sensitized to paranuclein but no more markedly than it does normal animals. It also fails to show specific intoxication in an animal that has been sensitized by the same substance. The amino acids, glutamic acid, and leucin, the principal components of their kind in casein, and in the same proportion therein present, likewise failed to show antigenic properties. They do not sensitize animals to milk intoxication or to intoxication by themselves, and likewise failed to produce precipitins in rabbits in a preliminary experiment. These experiments are regarded as a fairly systematic analysis of the antigenic properties of split products of a single protein. They are analogous to, though less complete than the work of Wells (6) on egg-white. They seem to present the additional advantage of dealing with what is probably the only protein certainly known chemically, and in its purest form. They serve, moreover, as an introduction to the following study of the antigenic properties of a combined protein. PMID:19867587

Effect of proteins from beef, pork, and turkey meat on plasma and liver lipids of rats compared with casein and soy protein.

PubMed

Brandsch, Corinna; Shukla, Anjali; Hirche, Frank; Stangl, Gabriele I; Eder, Klaus

2006-01-01

We assessed the effect of dietary proteins isolated from beef, pork, and turkey meat on concentrations of cholesterol and triacylglycerols in plasma, lipoproteins, and liver and the composition of the microsomal membrane (fatty acids, phosphatidylcholine/phosphatidylethanolamine ratio) compared with that of casein and soy protein in rats. Five groups of 12 rats each were fed semisynthetic diets for 20 d that contained 200 g/kg of proteins isolated from beef, pork, or turkey meat or, as controls, casein or soy protein. Rats fed beef, pork, or turkey proteins did not differ in cholesterol concentrations of plasma, lipoproteins, and liver and in composition of microsomal membrane from rats fed the casein diet. All groups fed a protein from an animal source had higher very low-density lipoprotein (VLDL) and liver cholesterol concentrations than did rats fed soy protein. However, rats fed pork protein had lower concentrations of triacylglycerols in liver, plasma, and VLDL and lower mRNA concentrations of sterol regulatory element binding protein-1 and glucose-6-phosphate dehydrogenase than did rats fed casein. However, concentrations of plasma and VLDL triacylglycerols in rats fed pork protein were not as low as those observed in rats fed soy protein. Proteins isolated from beef, pork, or turkey meat do not differ from casein in their effects on cholesterol metabolism. Pork protein decreases plasma triacylglycerol concentrations compared with casein but not compared with soy protein. The triacylglycerol-lowering effect of pork protein compared with casein is suggested to be caused by decreased hepatic fatty acid synthesis.

Relative efficacy of casein or soya protein combined with palm or safflower-seed oil on hyperuricaemia in rats.

PubMed

Lo, Hui-Chen; Wang, Yao-Horng; Chiou, Hue-Ying; Lai, Shan-Hu; Yang, Yu

2010-07-01

Diets that ameliorate the adverse effects of uric acid (UA) on renal damage deserve attention. The effects of casein or soya protein combined with palm or safflower-seed oil on various serum parameters and renal histology were investigated on hyperuricaemic rats. Male Wistar rats administered with oxonic acid and UA to induce hyperuricaemia were fed with casein or soya protein plus palm- or safflower-seed oil-supplemented diets. Normal rats and hyperuricaemic rats with or without allopurinol treatment (150 mg/l in drinking water) were fed with casein plus maize oil-supplemented diets. After 8 weeks, allopurinol treatment and soya protein plus safflower-seed oil-supplemented diet significantly decreased serum UA in hyperuricaemic rats (one-way ANOVA; P < 0.05). In addition, soya protein and casein attenuated hyperuricaemia-induced decreases in serum albumin and insulin, respectively (two-way ANOVA; P < 0.05). Safflower-seed oil significantly decreased serum TAG and UA, whereas palm oil significantly increased serum cholesterol, TAG, blood urea N and creatinine. However, soya protein significantly decreased renal NO and nitrotyrosine and palm oil significantly decreased renal nitrotyrosine, TNF-alpha and interferon-gamma and increased renal transforming growth factor-beta. Casein with safflower-seed oil significantly attenuated renal tubulointerstitial nephritis, crystals and fibrosis. Comparing casein v. soya protein combined with palm or safflower-seed oil, the results support that casein with safflower-seed oil may be effective in attenuating hyperuricaemia-associated renal damage, while soya protein with safflower-seed oil may be beneficial in lowering serum UA and TAG.

Influence of high glycine diets on the activity of glycine-catabolizing enzymes and on glycine catabolism in rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Petzke, K.J.; Albrecht, V.; Przybilski, H.

1986-05-01

Male albino rats were adapted to isocaloric purified diets that differed mainly in their glycine and casein contents. Controls received a 30% casein diet. In experimental diets gelatin or gelatin hydrolysate was substituted for half of the 30% casein. An additional group was fed a glycine-supplemented diet, which corresponded in glycine level to the gelatin diet but in which the protein level was nearly the same as that of the casein control diet. Another group received a 15% casein diet. Rat liver glycine cleavage system, serine hydroxymethyltransferase and serine dehydratase activities were measured. /sup 14/CO/sub 2/ production from the catabolismmore » of /sup 14/C-labeled glycine was measured in vivo and in vitro (from isolated hepatocytes). Serine dehydratase and glycine cleavage system activities were higher in animals fed 30% casein diets than in those fed 15% casein diets. Serine hydroxymethyltransferase activity of the cytosolic and mitochondrial fractions was highest when a high glycine diet (glycine administered as pure, protein bound in gelatin or peptide bound in gelatin hydrolysate) was fed. /sup 14/CO/sub 2/ formation from (1-/sup 14/C)- and (2-/sup 14/C)glycine both in vivo and in isolated hepatocytes was higher when a high glycine diet was fed than when a casein diet was fed. These results suggest that glycine catabolism is dependent on and adaptable to the glycine content of the diet. Serine hydroxymethyltransferase appears to play a major role in the regulation of glycine degradation via serine and pyruvate.« less

Establishment and characterization of a dairy goat mammary epithelial cell line with human telomerase (hT-MECs).

PubMed

Shi, Huaiping; Shi, Hengbo; Luo, Jun; Wang, Wei; Haile, Abiel B; Xu, Huifen; Li, Jun

2014-07-01

Although research on dairy goat mammary gland have referred extensively to molecular mechanisms, research on lines of dairy goat mammary epithelial cells (MECs) are still rare. This paper sought to establish an immortal MEC line by stable transfection of human telomerase. MECs from a lactating (45 days post-parturition) Xinong Saanen dairy goat were cultured purely and subsequently transfected with a plasmid carrying the sequence of human telomerase. Immortalized MECs by human telomerase (hT-MECs) exhibited a typical cobblestone morphology and activity and expression levels of telomerase resembled that of MCF-7 cells. hT-MECs on passage 42 grew vigorously and 'S' sigmoid curves of growth were observed. Moreover, hT-MECs maintained a normal chromosome modal number of 2n=60, keratin 8 and epithelial membrane antigen (EMA) were evidently expressed, and beta-casein protein was synthesized and secreted. Beta-casein expression was enhanced by prolactin (P<0.05). Lipid droplets were found in hT-MECs, and messenger RNA levels of PPARG, SREBP, FASN, ACC and SCD in hT-MECs (passage 40) were similar to MECs (passage 7). In conclusion, the obtained hT-MEC line retained a normal morphology, growth characteristics, cytogenetics and secretory characteristics as primary MECs. Hence, it can be a representative model cell line, for molecular and functional analysis, of dairy goat MECs for an extended period of time. © 2014 Japanese Society of Animal Science.

Casein hydrolysate augments antimicrobial and antioxidative efficacy of cheddar whey based edible coating of retail-cut beefsteak

USDA-ARS?s Scientific Manuscript database

Hydrolysis of casein using chymotrypsin results in the formation of polypeptides (casein hydrolyzate, CH) with a hydrophobic aromatic amino acid on one end of the chain because the enzyme selectively cleaves the adjacent peptide-bond. Due to resonance of the aromatic micro-domain, thiols become redo...

Binding analysis for interaction of diacetylcurcumin with β-casein nanoparticles by using fluorescence spectroscopy and molecular docking calculations

NASA Astrophysics Data System (ADS)

Mehranfar, Fahimeh; Bordbar, Abdol-Khalegh; Fani, Najme; Keyhanfar, Mehrnaz

2013-11-01

The interaction of diacetylcurcumin (DAC), as a novel synthetic derivative of curcumin, with bovine β-casein (an abundant milk protein that is highly amphiphilic and self assembles into stable micellar nanoparticles in aqueous solution) was investigated using fluorescence quenching experiments, Forster energy transfer measurements and molecular docking calculations. The fluorescence quenching measurements revealed the presence of a single binding site on β-casein for DAC with the binding constant value equals to (4.40 ± 0.03) × 104 M-1. Forster energy transfer measurements suggested that the distance between bound DAC and Trp143 residue is higher than the respective critical distance, hence, the static quenching is more likely responsible for fluorescence quenching other than the mechanism of non-radiative energy transfer. Our results from molecular docking calculations indicated that binding of DAC to β-casein predominantly occurred through hydrophobic contacts in the hydrophobic core of protein. Additionally, in vitro investigation of the cytotoxicity of free DAC and DAC-β-casein complex in human breast cancer cell line MCF7 revealed the higher cytotoxic effect of DAC-β-casein complex.

Initial stage of cheese production: a molecular modeling study of bovine and camel chymosin complexed with peptides from the chymosin-sensitive region of κ-casein.

PubMed

Sørensen, Jesper; Palmer, David S; Qvist, Karsten Bruun; Schiøtt, Birgit

2011-05-25

Bovine chymosin has long been the preferred enzyme used to coagulate cow's milk, in the initial stage of cheese production, during which it cleaves a specific bond in the milk protein κ-casein. Recently, camel chymosin has been shown to have a 70% higher clotting activity toward cow's milk and, moreover, to cleave κ-casein more selectively. Bovine chymosin, on the other hand, is a poor clotting agent toward camel's milk. This paper reports a molecular modeling study aimed at understanding this disparity, based on homology modeling and molecular dynamics simulations using peptide fragments of κ-casein from cow and camel in both bovine and camel chymosin. The results show that the complex between bovine chymosin and the fragment of camel κ-casein is indeed less stable in the binding pocket. The results also indicate that this in part may be due to charge repulsion between a lysine residue in bovine chymosin and an arginine residue in the P4 position of camel κ-casein.

THE RELATIVE EFFECTS OF PROTEIN, CHOLINE, AND METHIONINE IN THE TREATMENT OF EXPERIMENTAL DIETARY CIRRHOSIS IN THE RAT

PubMed Central

Plough, Irvin C.; Patek, Arthur J.; Bevans, Margaret

1952-01-01

Cirrhosis of the liver was produced in rats by feeding a diet low in protein (4 per cent casein) and deficient in lipotropic factors. The degree of liver cirrhosis was determined from specimens obtained at biopsy. Comparable groups of animals then were treated with diets containing 4 per cent casein and 30 per cent casein. The 4 per cent casein diets were supplemented with choline and methionine; the 30 per cent casein diets were fed with and without added choline. On supplementing the low protein diet with choline and methionine the animals remained feeble, their growth remained stunted, and their livers showed signs of progressive cirrhosis. In contrast, animals fed the higher protein diet (with or without added choline) grew normally, and their livers showed signs indicating arrest and regression of the disease process. These studies suggest that the feeding of high protein (30 per cent casein) diets to rats with nutritional cirrhosis produces reparative effects greater than those attributable to the supplements choline and methionine. PMID:14955576

Effects of peptides derived from dietary proteins on mucus secretion in rat jejunum.

PubMed

Claustre, Jean; Toumi, Férial; Trompette, Aurélien; Jourdan, Gérard; Guignard, Henri; Chayvialle, Jean Alain; Plaisancié, Pascale

2002-09-01

The hypothesis that dietary proteins or their hydrolysates may regulate intestinal mucin discharge was investigated in the isolated vascularly perfused rat jejunum using an enzyme-linked immunosorbent assay for rat intestinal mucins. On luminal administration, casein hydrolysate [0.05-5% (wt/vol)] stimulated mucin secretion in rat jejunum (maximal response at 417% of controls). Lactalbumin hydrolysate (5%) also evoked mucin discharge. In contrast, casein, and a mixture of amino acids was without effect. Chicken egg albumin and its hydrolysate or meat hydrolysate also did not modify mucin release. Interestingly, casein hydrolysate-induced mucin secretion was abolished by intra-arterial TTX or naloxone (an opioid antagonist). beta-Casomorphin-7, an opioid peptide released from beta-casein on milk ingestion, induced a strong mucin secretion (response at 563% of controls) that was inhibited by naloxone. Intra-arterial beta-casomorphin-7 also markedly increased mucin secretion (410% of controls). In conclusion, two enzymatic milk protein hydrolysates (casein and lactalbumin hydrolysates) and beta-casomorphin-7, specifically, induced mucin release in rat jejunum. The casein hydrolysate-induced mucin secretion is triggered by a neural pathway and mediated by opioid receptor activation.

Milk β-casein as a vehicle for delivery of bis(indolyl)methane: Spectroscopy and molecular docking studies

NASA Astrophysics Data System (ADS)

Dezhampanah, Hamid; Esmaili, Masoomeh; Khorshidi, Alireza

2017-05-01

The interaction of bis(indolyl)methane with bovine milk β-casein was investigated using spectroscopy and molecular docking studies at different temperatures (25-37 °C). The circular dichroism and Fourier transform infrared spectroscopic data demonstrated that β-casein interacts with BIM molecule mainly via both the hydrophobic and hydrophilic interactions with a minor change in the secondary structure of β-casein. The fluorescence quenching measurements revealed that the presence of a single binding site on β-casein for BIM with the binding constant value of ∼104 M-1. The negative values of entropy and enthalpy changes confirm the predominate role of hydrogen binding and van der Waals interactions in the binding process. Fӧrster energy transfer measurement suggested that the distance between bound BIM and Trp residue is higher than the respective critical distance. Hence, the static quenching is more likely responsible for the fluorescence quenching rather than the mechanism of non-radiative. Docking study showed that BIM molecule forms three hydrogen bonds and several van der Waals contacts with β-casein.

Effect of microfluidization on casein micelle size of bovine milk

NASA Astrophysics Data System (ADS)

Sinaga, H.; Deeth, H.; Bhandari, B.

2018-02-01

The properties of milk are likely to be dependent on the casein micelle size, and various processing technologies produce particular change in the average size of casein micelles. The main objective of this study was to manipulate casein micelle size by subjecting milk to microfluidizer. The experiment was performed as a complete block randomised design with three replications. The sample was passed through the microfluidizer at the set pressure of 83, 97, 112 and 126 MPa for one, two, three, four, five and six cycles, except for the 112 MPa. The results showed that microfluidized milk has smaller size by 3% with pressure up to 126 MPa. However, at each pressure, no further reduction was observed after increasing the passed up to 6 cycles. Although the average casein micelle size was similar, elevating pressure resulted in narrower size distribution. In contrast, increasing the number of cycles had little effect on casein micelle distribution. The finding from this study can be applied for future work to characterize the fundamental and functional properties of the treated milk.

Soy Glycinin Contains a Functional Inhibitory Sequence against Muscle-Atrophy-Associated Ubiquitin Ligase Cbl-b

PubMed Central

Yama, Tomonari; Ochi, Arisa; Suto, Takuro; Hirasaka, Katsuya; Teshima-Kondo, Shigetada; Okumura, Yuushi; Oarada, Motoko; Choi, Inho; Mukai, Rie; Terao, Junji

2013-01-01

Background. Unloading stress induces skeletal muscle atrophy. We have reported that Cbl-b ubiquitin ligase is a master regulator of unloading-associated muscle atrophy. The present study was designed to elucidate whether dietary soy glycinin protein prevents denervation-mediated muscle atrophy, based on the presence of inhibitory peptides against Cbl-b ubiquitin ligase in soy glycinin protein. Methods. Mice were fed either 20% casein diet, 20% soy protein isolate diet, 10% glycinin diet containing 10% casein, or 20% glycinin diet. One week later, the right sciatic nerve was cut. The wet weight, cross sectional area (CSA), IGF-1 signaling, and atrogene expression in hindlimb muscles were examined at 1, 3, 3.5, or 4 days after denervation. Results. 20% soy glycinin diet significantly prevented denervation-induced decreases in muscle wet weight and myofiber CSA. Furthermore, dietary soy protein inhibited denervation-induced ubiquitination and degradation of IRS-1 in tibialis anterior muscle. Dietary soy glycinin partially suppressed the denervation-mediated expression of atrogenes, such as MAFbx/atrogin-1 and MuRF-1, through the protection of IGF-1 signaling estimated by phosphorylation of Akt-1. Conclusions. Soy glycinin contains a functional inhibitory sequence against muscle-atrophy-associated ubiquitin ligase Cbl-b. Dietary soy glycinin protein significantly prevented muscle atrophy after denervation in mice. PMID:23762056

Effects of milk proteins on sperm binding to the zona pellucida and intracellular Ca(2+) concentration in stallion sperm.

PubMed

Coutinho da Silva, Marco A; Seidel, George E; Squires, Edward L; Graham, James K; Carnevale, Elaine M

2014-11-10

Objectives were to determine the effects of extracellular Ca(2+) and milk proteins on intracellular Ca(2+) concentrations in stallion sperm; and to determine the effects of single caseins on sperm binding to the zona pellucida (ZP). In Experiment I, sperm were incubated in media containing 2 or 4mM Ca(2+) and intracellular Ca(2+) concentration was determined after ionomycin treatment and long-term incubation (3h). Extracellular Ca(2+) concentrations (2 compared with 4mM) did not affect baseline intracellular Ca(2+) concentration of sperm. However, incubating sperm in a medium containing 4 compared with 2mM Ca(2+) resulted in greater (P<0.05) influx of Ca(2+) into sperm. In Experiment II, sperm incubated in media containing 1mg/mL of native phosphocaseinate (NP) or sodium caseinate (SC) showed similar baseline intracellular Ca(2+) and influx of Ca(2+) than control (TALP). In Experiment III, sperm-ZP binding assays were performed in TALP medium containing: no additions (TALP); 1mg/mL SC; 1 or 3mg/mL of α-casein; 1 or 3mg/mL of β-casein; and 1 or 3mg/mL of κ-casein. The number of stallion sperm bound to bovine ZP was greatest (P<0.05) when SC was used. Co-incubation in media containing single caseins (α-, β- or κ-casein) resulted in similar results to TALP; however, a dose effect (P<0.05) was observed for β- and κ-caseins. In conclusion, extracellular Ca(2+) concentration and milk proteins did not affect baseline intracellular calcium in stallion sperm. It appears that β- and κ-caseins may be responsible for enhancing sperm binding to ZP, but the mechanism remains unknown. Copyright © 2014 Elsevier B.V. All rights reserved.

Allergenicity of casein containing chalk in milk allergic schoolchildren.

PubMed

Larramendi, Carlos H; Marco, Francisco M; Llombart, Mónica; de la Vega, Ana; Chiner, Eusebi; García-Abujeta, José Luis; Sempere, José Miguel

2013-05-01

Nondietary exposure to milk proteins may be a risk for children who do not outgrow milk allergy by school age. To study the allergenicity of casein containing chalk. A 6-year-old, milk allergic child developed asthma and rhinoconjunctivitis while in school. The suspected cause was dust-free chalk containing casein. To study the relationship of dust-free chalk containing casein with asthma and rhinoconjunctivitis, 13 additional milk allergic patients were studied: 3 school-aged children, 8 preschool-aged infants, and 2 children with outgrown milk allergy. Skin tests and/or specific IgE with chalk and casein were performed. A chalk use test was performed in older children. Milk allergens contained in chalk were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblot, and IgE inhibition experiments. All school-aged, milk allergic children were exposed to chalk and reported symptoms attributed to chalk exposure. The skin test result to chalk was positive in 5 of 12 cases, and the specific IgE test result was positive in all 12 study participants in which it was performed. Casein strongly inhibited the binding of IgE to chalk. Chalk sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed proteins with molecular weight similar to caseins. Immunoblot demonstrated strong binding of IgE to chalk in a blurred pattern and a band at 30 kDa, inhibited by casein. The chalk challenge test result was positive in 2 school-age children who had a positive skin test result to chalk. Their symptoms improved after avoidance of chalk in the school. In 2 other cases in which the challenge test result was negative, chalk was reintroduced without problems. Inhalation of chalk dust containing casein can induce asthma symptoms in milk allergic patients. Hidden and nondietary sources of exposure should always be considered in food allergic patients. Copyright © 2013 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Analysis of DNA methylation of maize in response to osmotic and salt stress based on methylation-sensitive amplified polymorphism.

PubMed

Tan, Ming-pu

2010-01-01

Water stress is known to alter cytosine methylation, which generally represses transcription. However, little is known about the role of methylation alteration in maize under osmotic stress. Here, methylation-sensitive amplified polymorphism (MSAP) was used to screen PEG- or NaCl-induced methylation alteration in maize seedlings. The sequences of 25 differentially amplified fragments relevant to stress were successfully obtained. Two stress-specific fragments from leaves, LP166 and LPS911, shown to be homologous to retrotransposon Gag-Pol protein genes, suggested that osmotic stress-induced methylation of retrotransposons. Three MSAP fragments, representing drought-induced or salt-induced methylation in leaves, were homologous to a maize aluminum-induced transporter. Besides these, heat shock protein HSP82, Poly [ADP-ribose] polymerase 2, Lipoxygenase, casein kinase (CK2), and dehydration-responsive element-binding (DREB) factor were also homologs of MSAP sequences from salt-treated roots. One MSAP fragment amplified from salt-treated roots, designated RS39, was homologous to the first intron of maize protein phosphatase 2C (zmPP2C), whereas - LS103, absent from salt-treated leaves, was homologous to maize glutathione S-transferases (zmGST). Expression analysis showed that salt-induced intron methylation of root zmPP2C significantly downregulated its expression, while salt-induced demethylation of leaf zmGST weakly upregulated its expression. The results suggested that salinity-induced methylation downregulated zmPP2C expression, a negative regulator of the stress response, while salinity-induced demethylation upregulated zmGST expression, a positive effecter of the stress response. Altered methylation, in response to stress, might also be involved in stress acclimation. Copyright 2009 Elsevier Masson SAS. All rights reserved.

Soya protein stimulates bile acid excretion by the liver and intestine through direct and indirect pathways influenced by the presence of dietary cholesterol.

PubMed

Arellano-Martínez, Gloria Leticia; Granados, Omar; Palacios-González, Berenice; Torres, Nimbe; Medina-Vera, Isabel; Tovar, Armando R

2014-06-28

Several studies using different animal models have demonstrated that the consumption of soya protein (SP) reduces serum cholesterol concentrations by increasing the excretion of bile acids (BA). However, the mechanism by which SP enhances BA excretion is not fully understood. Therefore, the aim of the present study was to determine whether the consumption of SP regulates the expression of key enzymes involved in hepatic BA synthesis and the transporters involved in reverse cholesterol transport (RCT) via fibroblast growth factor 15 (FGF15) and/or small heterodimer protein (SHP) in rats. To achieve this aim, four groups of rats were fed experimental diets containing 20 % casein (C) or SP with or without the addition of 0·2 % cholesterol and the expression of hepatic genes involved in BA synthesis and the ileal and hepatic RCT was measured. Rats fed the SP diet had higher concentrations of ileal FGF15 and hepatic FGF15 receptor (FGFR4) and increased expression of SHP and liver receptor homolog 1 (LRH1) than those fed the C diet; as a result, the excretion of faecal BA was greater. The addition of cholesterol to the diet repressed the protein abundance of FGF15 and FGFR4; however, SP increased the expression of SHP and LRH1 to a lesser extent. Nonetheless, the expression of ABCG5/8 was increased in the intestine of rats fed the SP diet, and the effect was enhanced by the addition of cholesterol to the diet. In conclusion, SP in the presence of cholesterol increases BA synthesis via the repressions of FGF15 and SHP and accelerates BA excretion to prevent cholesterol overload in the enterocytes by increasing RCT.

Milk yield and genomewide expression profiling in the mammary gland of beef primiparous cows in response to the dietary management during the pre- and postweaning periods.

PubMed

Dervishi, E; Blanco, M; Rodríguez-Sánchez, J A; Sanz, A; Calvo, J H; Casasús, I

2017-10-01

Accelerated growth programs during prepubertal periods have been promoted to advance the first calving of beef heifers. The objectives of the present study were to evaluate nutrition-induced changes on first lactation milk yield and composition and on gene expression of the mammary gland in Parda de Montaña primiparous cows. Female calves ( = 16) were involved in a 2 × 2 factorial experiment. In the preweaning period (PRE-W; 0-6 mo), female calves were either fed a creep feed supplement (Creep) or fed only their dam's milk (Control). In the postweaning period (POST-W; 6-15 mo), heifers received either a high-energy diet (91.7 MJ/d) or a moderate-energy diet (79.3 MJ/d). All the heifers were managed together from breeding (15 mo) to the end of their first lactation (32 mo). Animal performance; milk production and quantity during the first lactation; plasma glucose, IGF-I, and leptin concentrations; and RNA samples from the mammary gland at the end of the first lactation of the primiparous cows (32 mo) were analyzed. The BW and ADG of the primiparous cow during its first lactation were not different among treatments; however, creep feeding during PRE-W reduced milk production ( < 0.01), milk CP, crude fat, lactose, nonfat solids, and casein content throughout lactation and increased somatic cell count in the third ( < 0.05) and fourth month of lactation ( < 0.10). The energy level during the POST-W had no effect on milk production and quality. Gene expression in the mammary gland was affected by the diet in the PRE-W and POST-W, with the PRE-W diet having the greatest impact. During the PRE-W, creep feeding resulted in upregulation of genes related to immune response and chemokine activity, suggesting that these animals might be in a compromised immune status. Therefore, this strategy would not be recommendable; meanwhile, increasing the energy level in the diet during the POST-W would be recommendable, because it had no deleterious effects on milk yield and composition.

[Not Available].

PubMed

Reyna, Nadia; Moreno-Rojas, Rafael; Mendoza, Laura; Parra, Karla; Linares, Sergia; Reyna, Eduardo; Cámara-Martos, Fernando

2016-02-16

It has been studied the effect of three kinds of supplements (whey, casein and maltodextrin, as control) in the regulation of food intake and satiety of 60 overweight women. After 10 weeks, significant differences (p < 0.001) were found with regard to reduction of weight, IMC, % fat and waist circumference in the whey group against casein and control groups. A higher decrease of energy intake (-383 kcal/day) was also found in women who ate whey supplements, while in the casein and control group the decrease was only -144 and -70 kcal/day respectively. Finally, satiety effect was more efficiently promoted by whey against casein and maltodextrins.

STUDIES ON THE FORMATION AND IONIZATION OF THE COMPOUNDS OF CASEIN WITH ALKALI

PubMed Central

Greenberg, David M.; Schmidt, Carl L. A.

1924-01-01

1. The results of conductivity experiments with alkali caseinate solutions are given and a graphical method of extrapolation, which gives a straight line, is described. The results of the conductivity experiments are shown to be in accord with the results of the previous transference experiments. 2. The change of conductivity of the alkali caseinate solutions with temperature is shown to follow a straight line relationship. 3. The high value of the mobility which was obtained for the casein ion and the high temperature gradient are discussed in relation to McBain's theory of colloidal electrolytes. PMID:19872136

Imaging and chemical surface analysis of biomolecular functionalization of monolithically integrated on silicon Mach-Zehnder interferometric immunosensors

NASA Astrophysics Data System (ADS)

Gajos, Katarzyna; Angelopoulou, Michailia; Petrou, Panagiota; Awsiuk, Kamil; Kakabakos, Sotirios; Haasnoot, Willem; Bernasik, Andrzej; Rysz, Jakub; Marzec, Mateusz M.; Misiakos, Konstantinos; Raptis, Ioannis; Budkowski, Andrzej

2016-11-01

Time-of-flight secondary ion mass spectrometry (imaging, micro-analysis) has been employed to evaluate biofunctionalization of the sensing arm areas of Mach-Zehnder interferometers monolithically integrated on silicon chips for the immunochemical (competitive) detection of bovine κ-casein in goat milk. Biosensor surfaces are examined after: modification with (3-aminopropyl)triethoxysilane, application of multiple overlapping spots of κ-casein solutions, blocking with 100-times diluted goat milk, and reaction with monoclonal mouse anti-κ-casein antibodies in blocking solution. The areas spotted with κ-casein solutions of different concentrations are examined and optimum concentration providing homogeneous coverage is determined. Coverage of biosensor surfaces with biomolecules after each of the sequential steps employed in immunodetection is also evaluated with TOF-SIMS, supplemented by Atomic force microscopy and X-ray photoelectron spectroscopy. Uniform molecular distributions are observed on the sensing arm areas after spotting with optimum κ-casein concentration, blocking and immunoreaction. The corresponding biomolecular compositions are determined with a Principal Component Analysis that distinguished between protein amino acids and milk glycerides, as well as between amino acids characteristic for Mabs and κ-casein, respectively. Use of the optimum conditions (κ-casein concentration) for functionalization of chips with arrays of ten Mach-Zehnder interferometers provided on-chips assays with dramatically improved both intra-chip response repeatability and assay detection sensitivity.

Does Milk Cause Constipation? A Crossover Dietary Trial

PubMed Central

Crowley, Elesa T.; Williams, Lauren T.; Roberts, Tim K.; Dunstan, Richard H.; Jones, Peter D.

2013-01-01

The aims of this study were to: (1) determine whether replacement of cow’s milk protein with soy resolves Chronic Functional Constipation (CFC); and (2) investigate the effects of cow’s milk β casein A1 and cow’s milk β casein A2 on CFC. Children diagnosed with CFC were recruited to one of two crossover trials: Trial 1 compared the effects of cow’s milk and soy milk; Trial 2 compared the effects of cow’s milk β casein A1 and cow’s milk β casein A2. Resolution of constipation was defined as greater than eight bowel motions during a two week intervention. Thirteen children (18 to 144 months) participated in Trial 1 (6 boys, 7 girls). Nine participants who completed the soy epoch all experienced resolution (p < 0.05). Thirty-nine children (21 to 144 months) participated in Trial 2 (25 boys, 14 girls). Resolution of constipation was highest during the washout epoch, 81%; followed by cow’s milk β casein A2, 79%; and cow’s milk β casein A1, 57%; however, the proportions did not differ statistically. The results of Trial 1 demonstrate an association between CFC and cow’s milk consumption but Trial 2 failed to show an effect from type of casein. Some other component in cow’s milk common to both A1 and A2 milk may be causing a problem in these susceptible children. PMID:23340316

The Contribution of Caseins to the Amino Acid Supply for Lactococcus lactis Depends on the Type of Cell Envelope Proteinase

PubMed Central

Flambard, Benedicte; Helinck, Sandra; Richard, Jean; Juillard, Vincent

1998-01-01

The ability of caseins to fulfill the amino acid requirements of Lactococcus lactis for growth was studied as a function of the type of cell envelope proteinase (PI versus PIII type). Two genetically engineered strains of L. lactis that differed only in the type of proteinase were grown in chemically defined media containing αs1-, β-, and κ-caseins (alone or in combination) as the sources of amino acids. Casein utilization resulted in limitation of the growth rate, and the extent of this limitation depended on the type of casein and proteinase. Adding different mixtures of essential amino acids to the growth medium made it possible to identify the nature of the limitation. This procedure also made it possible to identify the amino acid deficiency which was growth rate limiting for L. lactis in milk (S. Helinck, J. Richard, and V. Juillard, Appl. Environ. Microbiol. 63:2124–2130, 1997) as a function of the type of proteinase. Our results were compared with results from previous in vitro experiments in which casein degradation by purified proteinases was examined. The results were in agreement only in the case of the PI-type proteinase. Therefore, our results bring into question the validity of the in vitro approach to identification of casein-derived peptides released by a PIII-type proteinase. PMID:9603805

Reduced protein carbonylation of cube steak and catfish fillet using antioxidative coatings containing cheddar whey, casein hydrolyzate and oolong tea extract

USDA-ARS?s Scientific Manuscript database

Hydrolysis of casein using chymotrypsin results in the formation of polypeptides (casein hydrolyzate, CH) with a hydrophobic aromatic amino acid on one end of the chain because the enzyme selectively cleaves the adjacent peptide-bond. Due to resonance of the aromatic micro-domain, thiols become redo...

Effects of dietary soy protein on skeletal muscle volume and strength in humans with various physical activities.

PubMed

Hashimoto, Rie; Sakai, Atsuko; Murayama, Masumi; Ochi, Arisa; Abe, Tomoki; Hirasaka, Katsuya; Ohno, Ayako; Teshima-Kondo, Shigetada; Yanagawa, Hiroaki; Yasui, Natsuo; Inatsugi, Mikiko; Doi, Daisuke; Takeda, Masanori; Mukai, Rie; Terao, Junji; Nikawa, Takeshi

2015-01-01

In recent years, the number of bedridden people is rapidly increasing due to aging or lack of exercise in Japan. This problem is becoming more serious, since there is no countermeasure against it. In the present study, we designed to investigate whether dietary proteins, especially soy, had beneficial effects on skeletal muscle in 59 volunteers with various physical activities. We subjected 59 volunteers with various physical activities to meal intervention examination. Persons with low and high physical activities were divided into two dietary groups, the casein diet group and the soy diet group. They ate daily meals supplemented with 7.8 g of powdered casein or soy protein isolate every day for 30 days. Bedridden patients in hospitals were further divided into three dietary groups: the no supplementation diet group, the casein diet group and the soy diet group. They were also subjected to a blood test, a urinalysis, magnetic resonance imaging analysis and muscle strength test of the knee before and after the meal intervention study. Thirty-day soy protein supplementation significantly increased skeletal muscle volume in participants with low physical activity, compared with 30-day casein protein supplementation. Both casein and soy protein supplementation increased the volume of quadriceps femoris muscle in bedridden patients. Consistently, soy protein significantly increased their extension power of the knee, compared with casein protein. Although casein protein increased skeletal muscle volume more than soy protein in bedridden patients, their muscle strength changes by soy protein supplementation were bigger than those by casein protein supplementation. The supplementation of soy protein would be one of the effective foods which prevent the skeletal muscle atrophy caused by immobilization or unloading.

Influence of casein as a percentage of true protein and protein level on color and texture of milks containing 1 and 2% fat.

PubMed

Misawa, Noriko; Barbano, David M; Drake, MaryAnne

2016-07-01

Combinations of fresh liquid microfiltration retentate of skim milk, ultrafiltered retentate and permeate produced from microfiltration permeate, cream, and dried lactose monohydrate were used to produce a matrix of 20 milks. The milks contained 5 levels of casein as a percentage of true protein of about 5, 25, 50, 75, and 80% and 4 levels of true protein of 3.0, 3.76, 4.34, and 5.0% with constant lactose percentage of 5%. The experiment was replicated twice and repeated for both 1 and 2% fat content. Hunter color measurements, relative viscosity, and fat globule size distribution were measured, and a trained panel documented appearance and texture attributes on all milks. Overall, casein as a percentage of true protein had stronger effects than level of true protein on Hunter L, a, b values, relative viscosity, and fat globule size when using fresh liquid micellar casein concentrates and milk serum protein concentrates produced by a combination of microfiltration and ultrafiltration. As casein as a percentage of true protein increased, the milks became more white (higher L value), less green (lower negative a value), and less yellow (lower b value). Relative viscosity increased and d(0.9) generally decreased with increasing casein as a percentage of true protein. Panelists perceived milks with increasing casein as a percentage of true protein as more white, more opaque, and less yellow. Panelists were able to detect increased throat cling and mouthcoating with increased casein as a percentage of true protein in 2% milks, even when differences in appearance among milks were masked. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Skeletal effect of casein and whey protein intake during catch-up growth in young male Sprague-Dawley rats.

PubMed

Masarwi, Majdi; Gabet, Yankel; Dolkart, Oleg; Brosh, Tamar; Shamir, Raanan; Phillip, Moshe; Gat-Yablonski, Galia

2016-07-01

The aim of the present study was to determine whether the type of protein ingested influences the efficiency of catch-up (CU) growth and bone quality in fast-growing male rats. Young male Sprague-Dawley rats were either fed ad libitum (controls) or subjected to 36 d of 40 % food restriction followed by 24 or 40 d of re-feeding with either standard rat chow or iso-energetic, iso-protein diets containing milk proteins - casein or whey. In terms of body weight, CU growth was incomplete in all study groups. Despite their similar food consumption, casein-re-fed rats had a significantly higher body weight and longer humerus than whey-re-fed rats in the long term. The height of the epiphyseal growth plate (EGP) in both casein and whey groups was greater than that of rats re-fed normal chow. Microcomputed tomography yielded significant differences in bone microstructure between the casein and whey groups, with the casein-re-fed animals having greater cortical thickness in both the short and long term in addition to a higher trabecular bone fraction in the short term, although this difference disappeared in the long term. Mechanical testing confirmed the greater bone strength in rats re-fed casein. Bone quality during CU growth significantly depends on the type of protein ingested. The higher EGP in the casein- and whey-re-fed rats suggests a better growth potential with milk-based diets. These results suggest that whey may lead to slower bone growth with reduced weight gain and, as such, may serve to circumvent long-term complications of CU growth.

Addition of sodium caseinate to skim milk increases nonsedimentable casein and causes significant changes in rennet-induced gelation, heat stability, and ethanol stability.

PubMed

Lin, Yingchen; Kelly, Alan L; O'Mahony, James A; Guinee, Timothy P

2017-02-01

The protein content of skim milk was increased from 3.3 to 4.1% (wt/wt) by the addition of a blend of skim milk powder and sodium caseinate (NaCas), in which the weight ratio of skim milk powder to NaCas was varied from 0.8:0.0 to 0.0:0.8. Addition of NaCas increased the levels of nonsedimentable casein (from ∼6 to 18% of total casein) and calcium (from ∼36 to 43% of total calcium) and reduced the turbidity of the fortified milk, to a degree depending on level of NaCas added. Rennet gelation was adversely affected by the addition of NaCas at 0.2% (wt/wt) and completely inhibited at NaCas ≥0.4% (wt/wt). Rennet-induced hydrolysis was not affected by added NaCas. The proportion of total casein that was nonsedimentable on centrifugation (3,000 × g, 1 h, 25°C) of the rennet-treated milk after incubation for 1 h at 31°C increased significantly on addition of NaCas at ≥0.4% (wt/wt). Heat stability in the pH range 6.7 to 7.2 and ethanol stability at pH 6.4 were enhanced by the addition of NaCas. It is suggested that the negative effect of NaCas on rennet gelation is due to the increase in nonsedimentable casein, which upon hydrolysis by chymosin forms into small nonsedimentable particles that physically come between, and impede the aggregation of, rennet-altered para-casein micelles, and thereby inhibit the development of a gel network. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Isolation and characterization of beta- and gamma-caseins from horse milk.

PubMed Central

Visser, S; Jenness, R; Mullin, R J

1982-01-01

Three groups of casein components were isolated from horse milk. Group I is almost insoluble at acid and neutral pH, and is rather heterogeneous on alkaline gels with or without sodium dodecyl sulphate. Group II shows strong similarity to beta-casein from other species, as concluded from its amino acid composition and its N- and C-terminal sequences. This group consists of five electrophoretically distinguishable forms, all containing ester phosphate groups but no carbohydrate. Group III is composed of C-terminal fragments of the beta-like (group II) fraction and probably arises from the action of a plasmin-like enzyme present in horse milk. It does not contain phosphate or carbohydrate. Homology of this group with bovine gamma-caseins is demonstrated. Both beta- and gamma-like caseins are more soluble at 4 degrees C than at room temperature. Images Fig. 1. Fig. 3. Fig. 5. PMID:6213224

Mapping of Epitopes Occurring in Bovine α(s1)-Casein Variants by Peptide Microarray Immunoassay.

PubMed

Lisson, Maria; Erhardt, Georg

2016-01-01

Immunoglobulin E epitope mapping of milk proteins reveals important information about their immunologic properties. Genetic variants of αS1-casein, one of the major allergens in bovine milk, are until now not considered when discussing the allergenic potential. Here we describe the complete procedure to assess the allergenicity of αS1-casein variants B and C, which are frequent in most breeds, starting from milk with identification and purification of casein variants by isoelectric focusing (IEF) and anion-exchange chromatography, followed by in vitro gastrointestinal digestion of the casein variants, identification of the resulting peptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), in silico analysis of the variant-specific peptides as allergenic epitopes, and determination of their IgE-binding properties by microarray immunoassay with cow's milk allergic human sera.

Apparent voluminosity of casein micelles determined by rheometry.

PubMed

Nöbel, Stefan; Weidendorfer, Konrad; Hinrichs, Jörg

2012-11-15

The voluminosity of casein micelles was studied by means of static rheometry. In concentrated casein micelle suspensions with fluid-like flow properties to random-close packing, the reduced viscosity was obtained and linked via the Krieger-Dougherty model of volume fraction effect. The temperature dependency of hydration was fitted in a wide temperature (5°C≤θ≤35°C) and mass fraction range (0.01≤w≤0.16). The results of our study suggested that the voluminosity of casein micelles decreased with increasing temperature and asymptotically reached a plateau (θ>30°C) as a consequence of the protein swelling and decreasing water immobilization. The obtained apparent voluminosity of native casein micelles dispersed in UF permeate was 5.0 ml g(-1) at 5°C, 4.1 ml g(-1) at 20°C, and 3.7 ml g(-1) at 35°C. Copyright © 2012 Elsevier Inc. All rights reserved.

Effect of high hydrostatic pressure and whey proteins on the disruption of casein micelle isolates.

PubMed

Harte, Federico M; Gurram, Subba Rao; Luedecke, Lloyd O; Swanson, Barry G; Barbosa-Cánovas, Gustavo V

2007-11-01

High hydrostatic pressure disruption of casein micelle isolates was studied by analytical ultracentrifugation and transmission electron microscopy. Casein micelles were isolated from skim milk and subjected to combinations of thermal treatment (85 degrees C, 20 min) and high hydrostatic pressure (up to 676 MPa) with and without whey protein added. High hydrostatic pressure promoted extensive disruption of the casein micelles in the 250 to 310 MPa pressure range. At pressures greater than 310 MPa no further disruption was observed. The addition of whey protein to casein micelle isolates protected the micelles from high hydrostatic pressure induced disruption only when the mix was thermally processed before pressure treatment. The more whey protein was added (up to 5 g/l) the more the protection against high hydrostatic pressure induced micelle disruption was observed in thermally treated samples subjected to 310 MPa.

Physicochemical properties of low sodium frankfurter with added walnut: effect of transglutaminase combined with caseinate, KCl and dietary fibre as salt replacers.

PubMed

Colmenero, F Jiménez; Ayo, M J; Carballo, J

2005-04-01

This study compares the effects of combinations of microbial transglutaminase (TGase) and various non-meat ingredients (caseinate, KCl and wheat fibre) used as salt replacers, with the effects of NaCl on the physicochemical properties (cooking loss, emulsion stability, texture and colour) of frankfurters with added walnuts. The combination of TGase with caseinate, KCl or fibre led to harder, springier and chewier (P<0.05) frankfurters with better water- and fat-binding properties (emulsion stability and cooking loss) (P<0.05) than those made with TGase only. Ranking of ingredient efficiency in combination with TGase showed that caseinate>KCl>fibre. Frankfurters with caseinate presented the highest lightness and the lowest redness values. Frankfurter with NaCl had a harder, springier and chewier gel/emulsion network with lower cooking loss than those NaCl free.

Aroma barrier properties of sodium caseinate-based films.

PubMed

Fabra, Maria José; Hambleton, Alicia; Talens, Pau; Debeaufort, Fréderic; Chiralt, Amparo; Voilley, Andrée

2008-05-01

The mass transport of six different aroma compounds (ethyl acetate, ethyl butyrate, ethyl hexanoate, 2-hexanone, 1-hexanol, and cis-3-hexenol) through sodium caseinate-based films with different oleic acid (OA)/beeswax (BW) ratio has been studied. OA is less efficient than BW in reducing aroma permeability, which can be attributed to its greater polarity. Control film (without lipid) and films prepared with 0:100 OA/BW ratio show the lowest permeability. OA involves a decrease in aroma barrier properties of the sodium caseinate-based films due to its plasticization ability. Preferential sorption and diffusion occurs through OA instead of caseinate matrix and/or BW. The efficiency of sodium caseinate-based films to retain or limit aroma compound transfers depend on the affinity of the volatile compound to the films, which relates physicochemical interaction between volatile compound and film. Specific interactions (aroma compound-hydrocolloid and aroma compound-lipid) induce structural changes during mass transfer.

alpha-Lactalbumin species variation, HAMLET formation, and tumor cell death.

PubMed

Pettersson, Jenny; Mossberg, Ann-Kristin; Svanborg, Catharina

2006-06-23

HAMLET (human alpha-lactalbumin made lethal to tumor cells) is a tumoricidal complex of apo alpha-lactalbumin and oleic acid, formed in casein after low pH treatment of human milk. This study examined if HAMLET-like complexes are present in casein from different species and if isolated alpha-lactalbumin from those species can form such complexes with oleic acid. Casein from human, bovine, equine, and porcine milk was separated by ion exchange chromatography and active complexes were only found in human casein. This was not explained by alpha-lactalbumin sequence variation, as purified bovine, equine, porcine, and caprine alpha-lactalbumins formed complexes with oleic acid with biological activity similar to HAMLET. We conclude that structural variation of alpha-lactalbumins does not preclude the formation of HAMLET-like complexes and that natural HAMLET formation in casein was unique to human milk, which also showed the highest oleic acid content.

Increased extracellular matrix density decreases MCF10A breast cell acinus formation in 3D culture conditions.

PubMed

Lance, Amanda; Yang, Chih-Chao; Swamydas, Muthulekha; Dean, Delphine; Deitch, Sandy; Burg, Karen J L; Dréau, Didier

2016-01-01

The extracellular matrix (ECM) contributes to the generation and dynamic of normal breast tissue, in particular to the generation of polarized acinar and ductal structures. In vitro 3D culture conditions, including variations in the composition of the ECM, have been shown to directly influence the formation and organization of acinus-like and duct-like structures. Furthermore, the density of the ECM appears to also play a role in the normal mammary tissue and tumour formation. Here we show that the density of the ECM directly influences the number, organization and function of breast acini. Briefly, non-malignant human breast MCF10A cells were incubated in increasing densities of a Matrigel®-collagen I matrix. Elastic moduli near and distant to the acinus structures were measured by atomic force microscopy, and the number of acinus structures was determined. Immunochemistry was used to investigate the expression levels of E-cadherin, laminin, matrix metalloproteinase-14 and ß-casein in MCF10A cells. The modulus of the ECM was significantly increased near the acinus structures and the number of acinus structures decreased with the increase in Matrigel-collagen I density. As evaluated by the expression of laminin, the organization of the acinus structures present was altered as the density of the ECM increased. Increases in both E-cadherin and MMP14 expression by MCF10A cells as ECM density increased were also observed. In contrast, MCF10A cells expressed lower ß-casein levels as the ECM density increased. Taken together, these observations highlight the key role of ECM density in modulating the number, organization and function of breast acini. Copyright © 2013 John Wiley & Sons, Ltd.

High casein kinase 1 epsilon levels are correlated with better prognosis in subsets of patients with breast cancer

PubMed Central

Lopez-Guerra, Jose Luis; Verdugo-Sivianes, Eva M.; Otero-Albiol, Daniel; Vieites, Begoña; Ortiz-Gordillo, Maria J.; De León, Jose M.; Praena-Fernandez, Juan M.; Marin, Juan J.; Carnero, Amancio

2015-01-01

Reliable biological markers that predict breast cancer (BC) outcomes after multidisciplinary therapy have not been fully elucidated. We investigated the association between casein kinase 1 epsilon (CK1ε) and the risk of recurrence in patients with BC. Using 168 available tumor samples from patients with BC treated with surgery +/− chemo(radio)therapy, we scored the CK1ε expression as high (≥1.5) or low (<1.5) using an immunohistochemical method. Kaplan-Meier analysis was performed to assess the risk of relapse, and Cox proportional hazards analyses were utilized to evaluate the effect of CK1ε expression on this risk. The median age at diagnosis was 60 years (range 35-96). A total of 58% of the patients underwent breast conservation surgery, while 42% underwent mastectomy. Adjuvant chemotherapy and radiation therapy were administered in 101 (60%) and 137 cases (82%), respectively. Relapse was observed in 24 patients (14%). Multivariate analysis found high expression of CK1ε to be associated with a statistically significant higher disease-free survival (DFS) in BC patients with wild-type p53 (Hazard ratio [HR] = 0.33; 95% CI, 0.12-0.91; P = 0.018) or poor histological differentiation ([HR] = 0.34; 95% CI, 0.12-0.94; P = 0.039) or in those without adjuvant chemotherapy ([HR] = 0.11; 95% CI, 0.01-0.97; P = 0.006). Our data indicate that CK1ε expression is associated with DFS in BC patients with wild-type p53 or poor histological differentiation or in those without adjuvant chemotherapy and thus may serve as a predictor of recurrence in these subsets of patients. PMID:26327509

Rapid chromatographic determination of caseins in milk with photometric and fluorimetric detection using a hydrophobic monolithic column.

PubMed

Ramírez-Palomino, P; Fernández-Romero, J M; Gómez-Hens, A

2014-01-01

Reverse-phase liquid chromatographic methods using a hydrophobic C18 monolithic column and on-line photometric and fluorimetric detection for the determination of the major casein (CN) proteins in milk are presented. The separation of αs1-CN, αs2-CN, β-CN and κ-CN was achieved in only five minutes. Fluorimetric detection enabled better analytical results than photometric detection. Thus, the dynamic ranges of the calibration graphs and detection limits obtained using fluorimetric detection were (mgmL(-)(1)): αs1-CN (0.74-10.0, 0.22), αs2-CN (0.15-10.0, 0.045), β-CN (0.68-10.0, 0.20) and κ-CN (0.21-10.0, 0.06). The analytical features of the photometric method, which does not allow the quantification of β-casein, were (mgmL(-)(1)): αs1-CN (1.5-9.0, 0.45), αs2-CN (1.4-10.0, 0.43) and κ-CN (0.4-9.0, 0.12). Precision data, expressed as relative standard deviation, ranged between 0.6% and 5.3% for the fluorimetric method and between 2.4% and 6.2% for the photometric method. Both methods were applied to the analysis of three different milk samples, obtaining recoveries in the ranges of 86.6-103.2% and 92.0-106.5% using fluorimetric and photometric detection, respectively. Copyright © 2013 Elsevier Ltd. All rights reserved.

PACSIN2 polymorphism influences TPMT activity and mercaptopurine-related gastrointestinal toxicity.

PubMed

Stocco, Gabriele; Yang, Wenjian; Crews, Kristine R; Thierfelder, William E; Decorti, Giuliana; Londero, Margherita; Franca, Raffaella; Rabusin, Marco; Valsecchi, Maria Grazia; Pei, Deqing; Cheng, Cheng; Paugh, Steven W; Ramsey, Laura B; Diouf, Barthelemy; McCorkle, Joseph Robert; Jones, Terreia S; Pui, Ching-Hon; Relling, Mary V; Evans, William E

2012-11-01

Treatment-related toxicity can be life-threatening and is the primary cause of interruption or discontinuation of chemotherapy for acute lymphoblastic leukemia (ALL), leading to an increased risk of relapse. Mercaptopurine is an essential component of continuation therapy in all ALL treatment protocols worldwide. Genetic polymorphisms in thiopurine S-methyltransferase (TPMT) are known to have a marked effect on mercaptopurine metabolism and toxicity; however, some patients with wild-type TPMT develop toxicity during mercaptopurine treatment for reasons that are not well understood. To identify additional genetic determinants of mercaptopurine toxicity, a genome-wide analysis was performed in a panel of human HapMap cell lines to identify trans-acting genes whose expression and/or single-nucleotide polymorphisms (SNPs) are related to TPMT activity, then validated in patients with ALL. The highest ranking gene with both mRNA expression and SNPs associated with TPMT activity in HapMap cell lines was protein kinase C and casein kinase substrate in neurons 2 (PACSIN2). The association of a PACSIN2 SNP (rs2413739) with TPMT activity was confirmed in patients and knock-down of PACSIN2 mRNA in human leukemia cells (NALM6) resulted in significantly lower TPMT activity. Moreover, this PACSIN2 SNP was significantly associated with the incidence of severe gastrointestinal (GI) toxicity during consolidation therapy containing mercaptopurine, and remained significant in a multivariate analysis including TPMT and SLCO1B1 as covariates, consistent with its influence on TPMT activity. The association with GI toxicity was also validated in a separate cohort of pediatric patients with ALL. These data indicate that polymorphism in PACSIN2 significantly modulates TPMT activity and influences the risk of GI toxicity associated with mercaptopurine therapy.

PACSIN2 polymorphism influences TPMT activity and mercaptopurine-related gastrointestinal toxicity

PubMed Central

Stocco, Gabriele; Yang, Wenjian; Crews, Kristine R.; Thierfelder, William E.; Decorti, Giuliana; Londero, Margherita; Franca, Raffaella; Rabusin, Marco; Valsecchi, Maria Grazia; Pei, Deqing; Cheng, Cheng; Paugh, Steven W.; Ramsey, Laura B.; Diouf, Barthelemy; McCorkle, Joseph Robert; Jones, Terreia S.; Pui, Ching-Hon; Relling, Mary V.; Evans, William E.

2012-01-01

Treatment-related toxicity can be life-threatening and is the primary cause of interruption or discontinuation of chemotherapy for acute lymphoblastic leukemia (ALL), leading to an increased risk of relapse. Mercaptopurine is an essential component of continuation therapy in all ALL treatment protocols worldwide. Genetic polymorphisms in thiopurine S-methyltransferase (TPMT) are known to have a marked effect on mercaptopurine metabolism and toxicity; however, some patients with wild-type TPMT develop toxicity during mercaptopurine treatment for reasons that are not well understood. To identify additional genetic determinants of mercaptopurine toxicity, a genome-wide analysis was performed in a panel of human HapMap cell lines to identify trans-acting genes whose expression and/or single-nucleotide polymorphisms (SNPs) are related to TPMT activity, then validated in patients with ALL. The highest ranking gene with both mRNA expression and SNPs associated with TPMT activity in HapMap cell lines was protein kinase C and casein kinase substrate in neurons 2 (PACSIN2). The association of a PACSIN2 SNP (rs2413739) with TPMT activity was confirmed in patients and knock-down of PACSIN2 mRNA in human leukemia cells (NALM6) resulted in significantly lower TPMT activity. Moreover, this PACSIN2 SNP was significantly associated with the incidence of severe gastrointestinal (GI) toxicity during consolidation therapy containing mercaptopurine, and remained significant in a multivariate analysis including TPMT and SLCO1B1 as covariates, consistent with its influence on TPMT activity. The association with GI toxicity was also validated in a separate cohort of pediatric patients with ALL. These data indicate that polymorphism in PACSIN2 significantly modulates TPMT activity and influences the risk of GI toxicity associated with mercaptopurine therapy. PMID:22846425

Dietary protein sources differentially affect microbiota, mTOR activity and transcription of mTOR signaling pathways in the small intestine

PubMed Central

Kar, Soumya K.; Jansman, Alfons J. M.; Benis, Nirupama; Ramiro-Garcia, Javier; Schokker, Dirkjan; Kruijt, Leo; Stolte, Ellen H.; Taverne-Thiele, Johanna J.; Smits, Mari A.; Wells, Jerry M.

2017-01-01

Dietary protein sources can have profound effects on host-microbe interactions in the gut that are critically important for immune resilience. However more knowledge is needed to assess the impact of different protein sources on gut and animal health. Thirty-six wildtype male C57BL/6J mice of 35 d age (n = 6/group; mean ± SEM body weight 21.9 ± 0.25 g) were randomly assigned to groups fed for four weeks with semi synthetic diets prepared with one of the following protein sources containing (300 g/kg as fed basis): soybean meal (SBM), casein, partially delactosed whey powder, spray dried plasma protein, wheat gluten meal and yellow meal worm. At the end of the experiment, mice were sacrificed to collect ileal tissue to acquire gene expression data, and mammalian (mechanistic) target of rapamycin (mTOR) activity, ileal digesta to study changes in microbiota and serum to measure cytokines and chemokines. By genome-wide transcriptome analysis, we identified fourteen high level regulatory genes that are strongly affected in SBM-fed mice compared to the other experimental groups. They mostly related to the mTOR pathway. In addition, an increased (P < 0.05) concentration of granulocyte colony-stimulating factor was observed in serum of SBM-fed mice compared to other dietary groups. Moreover, by 16S rRNA sequencing, we observed that SBM-fed mice had higher (P < 0.05) abundances of Bacteroidales family S24-7, compared to the other dietary groups. We showed that measurements of genome-wide expression and microbiota composition in the mouse ileum reveal divergent responses to diets containing different protein sources, in particular for a diet based on SBM. PMID:29149221

Comparative analysis and molecular characterization of a gene BANF1 encoded a DNA-binding protein during mitosis from the Giant Panda and Black Bear.

PubMed

Zeng, Yichun; Hou, Yi-Ling; Ding, Xiang; Hou, Wan-Ru; Li, Jian

2014-01-01

Barrier to autointegration factor 1 (BANF1) is a DNA-binding protein found in the nucleus and cytoplasm of eukaryotic cells that functions to establish nuclear architecture during mitosis. The cDNA and the genomic sequence of BANF1 were cloned from the Giant Panda (Ailuropoda melanoleuca) and Black Bear (Ursus thibetanus mupinensis) using RT-PCR technology and Touchdown-PCR, respectively. The cDNA of the BANF1 cloned from Giant Panda and Black Bear is 297 bp in size, containing an open reading frame of 270 bp encoding 89 amino acids. The length of the genomic sequence from Giant Panda is 521 bp, from Black Bear is 536 bp, which were found both to possess 2 exons. Alignment analysis indicated that the nucleotide sequence and the deduced amino acid sequence are highly conserved to some mammalian species studied. Topology prediction showed there is one Protein kinase C phosphorylation site, one Casein kinase II phosphorylation site, one Tyrosine kinase phosphorylation site, one N-myristoylation site, and one Amidation site in the BANF1 protein of the Giant Panda, and there is one Protein kinase C phosphorylation site, one Tyrosine kinase phosphorylation site, one N-myristoylation site, and one Amidation site in the BANF1 protein of the Black Bear. The BANF1 gene can be readily expressed in E. coli. Results showed that the protein BANF1 fusion with the N-terminally His-tagged form gave rise to the accumulation of an expected 14 kD polypeptide that formed inclusion bodies. The expression products obtained could be used to purify the proteins and study their function further.

Cross-linking of bovine and caprine caseins by microbial transglutaminase and their use as microencapsulating agents for n-3 fatty acids

USDA-ARS?s Scientific Manuscript database

Bovine and caprine caseins were cross-linked with microbial transglutaminase (mTG). The mTG-cross-linked bovine or caprine casein dispersion, mixed with 14.5% maltodextrin (DE = 40), was used to prepare emulsions with 10.5% algae oil. Oxidative stability of emulsions was evaluated by peroxide valu...

RNA-sequencing data analysis of uterus in ovariectomized rats fed with soy protein isolate,17B-estradiol and casein

USDA-ARS?s Scientific Manuscript database

This data file describes the bioinformatics analysis of uterine RNA-seq data comparing genome wide effects of feeding soy protein isolate compared to casein to ovariectomized female rats age 64 days relative to treatment of casein fed rats with 5 ug/kg/d estradiol and relative to rats treated with e...

Contribution of Molecular Allergen Analysis in Diagnosis of Milk Allergy.

PubMed

Bartuzi, Zbigniew; Cocco, Renata Rodrigues; Muraro, Antonella; Nowak-Węgrzyn, Anna

2017-07-01

We sought to describe the available evidence supporting the utilization of the molecular allergen analysis (MAA) for diagnosis and management of cow milk protein allergy (CMPA). Cow milk proteins are among the most common food allergens in IgE- and non-IgE-mediated food allergic disorders in children. Most individuals with CMPA are sensitized to both caseins and whey proteins. Caseins are more resistant to high temperatures compared to whey proteins. MAA is not superior to the conventional diagnostic tests based on the whole allergen extracts for diagnosis of CMPA. However, MAA can be useful in diagnosing tolerance to extensively heated milk proteins in baked foods. Children with CMPA and high levels of casein IgE are less likely to tolerate baked milk compared to children with low levels of casein IgE. Specific IgE-binding patterns to casein and betalactoglobulin peptides may predict the natural course of CMPA and differentiate subjects who are more likely to develop CMPA at a younger age versus those with a more persistent CMPA. Specific IgE-binding patterns to casein and beta-lactoglobulin peptides may also predict response to milk OITand identify patientsmost likely to benefit fromOIT.

Conformational transition of κ-casein in micellar environment: Insight from the tryptophan fluorescence

NASA Astrophysics Data System (ADS)

Mishra, Smruti; Meher, Geetanjali; Chakraborty, Hirak

2017-11-01

Intrinsically disordered proteins (IDPs) are under intense analysis due to their structural flexibility and importance in biological functions. Minuscule modulation in the microenvironment induces significant conformational changes in IDPs, and these non-native conformations of the IDPs often induce aggregation and cause cell death. Changes in the membrane composition often change the microenvironment, which promote conformational change and aggregation of IDPs. κ-Casein, an important milk protein, belongs to the class of IDPs containing net negative charges. In this present work, we have studied the interaction of κ-casein with cetyltrimethyl ammonium bromide (CTAB), a positively charged surfactant, utilizing various steady state fluorescence, time-resolved fluorescence and circular dichroism spectroscopy. Our results clearly indicate that κ-casein undergoes at least two conformational transitions in presence of various concentrations of CTAB. The intrinsically disordered κ-casein assumes a partially folded conformation at lower concentration of CTAB, which adopts an unstructured conformation at higher concentration of CTAB. The partially folded conformation of κ-casein at a lower CTAB concentration might be induced by the favorable electrostatic interaction between the positively charged surfactant headgroup and net negative charges of the protein, whereas surfactant nature of CTAB is being pronounced at higher concentration of CTAB.

High pressure homogenization to improve the stability of casein - hydroxypropyl cellulose aqueous systems.

PubMed

Ye, Ran; Harte, Federico

2014-03-01

The effect of high pressure homogenization on the improvement of the stability hydroxypropyl cellulose (HPC) and micellar casein was investigated. HPC with two molecular weights (80 and 1150 kDa) and micellar casein were mixed in water to a concentration leading to phase separation (0.45% w/v HPC and 3% w/v casein) and immediately subjected to high pressure homogenization ranging from 0 to 300 MPa, in 100 MPa increments. The various dispersions were evaluated for stability, particle size, turbidity, protein content, and viscosity over a period of two weeks and Scanning Transmission Electron Microscopy (STEM) at the end of the storage period. The stability of casein-HPC complexes was enhanced with the increasing homogenization pressure, especially for the complex containing high molecular weight HPC. The apparent particle size of complexes was reduced from ~200nm to ~130nm when using 300 MPa, corresponding to the sharp decrease of absorbance when compared to the non-homogenized controls. High pressure homogenization reduced the viscosity of HPC-casein complexes regardless of the molecular weight of HPC and STEM imagines revealed aggregates consistent with nano-scale protein polysaccharide interactions.

High pressure homogenization to improve the stability of casein - hydroxypropyl cellulose aqueous systems

PubMed Central

Ye, Ran; Harte, Federico

2013-01-01

The effect of high pressure homogenization on the improvement of the stability hydroxypropyl cellulose (HPC) and micellar casein was investigated. HPC with two molecular weights (80 and 1150 kDa) and micellar casein were mixed in water to a concentration leading to phase separation (0.45% w/v HPC and 3% w/v casein) and immediately subjected to high pressure homogenization ranging from 0 to 300 MPa, in 100 MPa increments. The various dispersions were evaluated for stability, particle size, turbidity, protein content, and viscosity over a period of two weeks and Scanning Transmission Electron Microscopy (STEM) at the end of the storage period. The stability of casein-HPC complexes was enhanced with the increasing homogenization pressure, especially for the complex containing high molecular weight HPC. The apparent particle size of complexes was reduced from ~200nm to ~130nm when using 300 MPa, corresponding to the sharp decrease of absorbance when compared to the non-homogenized controls. High pressure homogenization reduced the viscosity of HPC-casein complexes regardless of the molecular weight of HPC and STEM imagines revealed aggregates consistent with nano-scale protein polysaccharide interactions. PMID:24159250

Optimization of vitamins A and D3 loading in re-assembled casein micelles and effect of loading on stability of vitamin D3 during storage.

PubMed

Loewen, Anisa; Chan, Benny; Li-Chan, Eunice C Y

2018-02-01

The objectives of this study were to apply response surface methodology to optimize fat-soluble vitamin loading in re-assembled casein micelles, and to evaluate vitamin D stability of dry formulations during ambient or accelerated storage and in fortified fluid skim milk stored under refrigeration. Optimal loading of vitamin A (1.46-1.48mg/100mgcasein) was found at 9.7mM phosphate, 5.5mM citrate and 30.0mM calcium, while optimal loading of vitamin D (1.38-1.46mg/100mg casein) was found at 4.9mM phosphate, 4.0mM citrate and 26.1mM calcium. In general, more vitamin D was retained in vitamin D-re-assembled casein micelles than control powders during storage, while vitamin D loss was not different for vitamin D-re-assembled casein micelles and control fortified milks after 21days of refrigerated storage with light exposure. In conclusion, re-assembled casein micelles with high loading efficiency show promise for improving vitamin D stability during dry storage. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Nuclear magnetic resonance study of the conformation and dynamics of beta-casein at the oil/water interface in emulsions.

PubMed Central

ter Beek, L C; Ketelaars, M; McCain, D C; Smulders, P E; Walstra, P; Hemminga, M A

1996-01-01

A (13)C and (31)P nuclear magnetic resonance (NMR) study has been carried out on beta-casein adsorbed at the interface of a tetradecane/water emulsion. (13)C NMR spectra show signals from the carbonyl, carboxyl, aromatic, and C alpha carbons in beta-casein, well resolved from solvent resonances. Only a small fraction of all carbon atoms in beta-casein contribute to detectable signals; intensity measurements show that the observable spectrum is derived from about 30 to 40 amino acid residues.(31)P NMR spectra show signals from the five phosphoserines on the hydrophilic N-terminal part of the protein. Analysis of T(1) relaxation times of these nuclei, using the model free approach for the spectral density function and the line shape of the alpha-carbon region, indicates that a large part of the protein is in a random coil conformation with restricted motion and a relatively long internal correlation time. The NMR results show that the conformation and dynamics of the N-terminal part of beta-casein are not strongly altered at the oil/water interface, as compared to beta-casein in micelle-like aggregates in aqueous solution. PMID:9172765

Effects of the environmental factors on the casein micelle structure studied by cryo transmission electron microscopy and small-angle x-ray scattering/ultrasmall-angle x-ray scattering

NASA Astrophysics Data System (ADS)

Marchin, Stéphane; Putaux, Jean-Luc; Pignon, Frédéric; Léonil, Joëlle

2007-01-01

Casein micelles are colloidal protein-calcium-transport complexes whose structure has not been unequivocally elucidated. This study used small-angle x-ray scattering (SAXS) and ultrasmall angle x-ray scattering (USAXS) as well as cryo transmission electron microscopy (cryo-TEM) to provide fine structural details on their structure. Cryo-TEM observations of native casein micelles fractionated by differential centrifugation showed that colloidal calcium phosphate appeared as nanoclusters with a diameter of about 2.5nm. They were uniformly distributed in a homogeneous tangled web of caseins and were primarily responsible for the intensity distribution in the SAXS profiles at the highest q vectors corresponding to the internal structure of the casein micelles. A specific demineralization of casein micelles by decreasing the pH from 6.7 to 5.2 resulted in a reduced granular aspect of the micelles observed by cryo-TEM and the existence of a characteristic point of inflection in SAXS profiles. This supports the hypothesis that the smaller substructures detected by SAXS are colloidal calcium phosphate nanoclusters rather than putative submicelles.

Effect of pH on turbidity, size, viscosity and the shape of sodium caseinate aggregates with light scattering and rheometry.

PubMed

Ghorbani Gorji, Sara; Ghorbani Gorji, Elham; Mohammadifar, Mohammad Amin

2015-03-01

The characterization of sodium caseinate solutions as a function of pH was determined using titration with HCL through turbidimetry in different concentrations (0.03 wt.%, 0.045 wt.%, 0.06 wt.%, 0.09 wt.%, 0.2 wt.%, and 0.3 wt.%). Additionally, the coupling of slow in situ acidification of the solution and rheometry was utilized to gain deeper insights into pH-induced structural transitions during the self assembly process and particle size distribution analysis have been used to determine the behavior of sodium caseinate solutions in different pHs. The formation of aggregates during the acidification process was clearly visualized using microscopy. Surprisingly the viscosity of sodium caseinate solution at pH 4.64 was maximum and decreased by lowering pH. Particle size analysis confirmed the onset of big aggregates on decreasing pH but further acidification led to formation of smaller aggregates. A small concentration effect on pI was seen where at sodium caseinate levels of 0.03 wt.% the pI occurred at 4.29, where at sodium caseinate levels of 0.30 wt.% pI value was 4.64.

Rheological behavior of high-concentration sodium caseinate dispersions.

PubMed

Loveday, Simon M; Rao, M Anandha; Creamer, Lawrence K; Singh, Harjinder

2010-03-01

Apparent viscosity and frequency sweep (G', G'') data for sodium caseinate dispersions with concentrations of approximately 18% to 40% w/w were obtained at 20 degrees C; colloidal glass behavior was exhibited by dispersions with concentration >or=23% w/w. The high concentrations were obtained by mixing frozen powdered buffer with sodium caseinate in boiling liquid nitrogen, and allowing the mixtures to thaw and hydrate at 4 degrees C. The low-temperature G'-G'' crossover seen in temperature scans between 60 and 5 degrees C was thought to indicate gelation. Temperature scans from 5 to 90 degrees C revealed gradual decrease in G' followed by plateau values. In contrast, G'' decreased gradually and did not reach plateau values. Increase in hydrophobicity of the sodium caseinate or a decrease in the effective volume fraction of its aggregates may have contributed to these phenomena. The gelation and end of softening temperatures of the dispersions increased with the concentration of sodium caseinate. From an Eldridge-Ferry plot, the enthalpy of softening was estimated to be 29.6 kJ mol(-1). The results of this study should be useful for creating new products with high concentrations of sodium caseinate.

Physicochemical characterization of native and modified sodium caseinate- Vitamin A complexes.

PubMed

Gupta, Chitra; Arora, Sumit; Syama, M A; Sharma, Apurva

2018-04-01

Native and modified sodium caseinate- Vitamin A complexes {Sodium caseinate- Vit A complex by stirring (NaCas-VA ST), succinylated sodium caseinate- Vit A complex by stirring (SNaCas-VA ST), reassembled sodium caseinate- Vit A complex (RNaCas-VA) and reassembled succinylated sodium caseinate- Vit A complex (RSNaCas-VA)} were prepared and characterized for their physicochemical characteristics e.g. particle size, zeta potential, turbidity analysis and tryptophan intensities which confirmed structural modification of both native (NaCas-VA ST) and modified (SNaCas-VA ST, RNaCas-VA and RSNaCas- VA) proteins upon complex formation with vitamin A. Binding of vitamin A to milk protein reduced the turbidity caused by vitamin A, however, the particle size and zeta potential of milk protein increased after complexation. Microstructure details of NaCas (spray dried) showed uniform spherical structure, however, other milk proteins and milk protein- Vit A complexes (freeze dried) showed broken glass and flaky structures. Tiny particles were observed on the surface of reassembled protein and reassembled protein- Vit A complexes. Binding of vitamin A to milk protein did not have an influence on the electrophoretic mobility and elution profile (RP-HPLC). Copyright © 2018 Elsevier Ltd. All rights reserved.

Interactions of dietary protein and carbohydrate determine blood sugar level and regulate nutrient selection in the insect Manduca sexta L.

PubMed

Thompson, S N; Redak, R A

2000-09-01

The non-homeostatic regulation of blood sugar concentration in the insect Manduca sexta L. was affected by nutritional status. Larvae maintained on diets lacking sucrose displayed low concentrations of trehalose, the blood sugar of insects, which varied from 5 to 15 mM with increasing dietary casein level between 12.5 and 75 g/l. These insects were glucogenic, as demonstrated by the selective 13C enrichment of trehalose synthesized from [3-13C]alanine, and de novo synthesis was the sole source of blood sugar. The distribution of 13C in glutamine established that following transamination of the 13C substituted substrate, [3-13C]pyruvate carboxylation rather than decarboxylation was the principal pathway of Pyr metabolism. The mean blood trehalose level was higher in insects maintained on diets with sucrose. At the lowest dietary casein level blood trehalose was approximately 50 mM, and declined to 20 mM at the highest casein level. Gluconeogenesis was detected in insects maintained on sucrose-free diets at the higher protein levels examined, but [3-13C]pyruvate decarboxylation and TCA cycle metabolism was the principal fate of [3-13C]alanine following transamination, and dietary carbohydrate was the principal source of glucose for trehalose synthesis. Feeding studies established a relationship between nutritional status, blood sugar level and dietary self-selection. Insects preconditioned by feeding on diets without sucrose had low blood sugar levels regardless of dietary casein level, and when subsequently given a choice between a sucrose diet or a casein diet, selected the former. Larvae preconditioned on a diet containing sucrose and the lowest level of casein had high blood sugar levels and subsequently selected the casein diet. Larvae maintained on the sucrose diet with the highest casein level had low blood sugar and self-selected the sucrose diet. When preconditioned on diets with sucrose and intermediate levels of casein, insects selected more equally between the sucrose and the casein diets. It is concluded that blood sugar level may be intimately involved in dietary self-selection by M. sexta larvae, and that in the absence of dietary carbohydrate, gluconeogenesis provides sufficient blood sugar to ensure that larvae choose a diet or diets that produce an optimal intake of dietary protein and carbohydrate.

Progesterone Receptor Scaffolding Function in Breast Cancer

DTIC Science & Technology

2012-10-01

phosphorylation is dependent on the serine- threonine kinase, ck2. Cumulatively, these data suggest that mutation of PR’s CD domain alters the program...liganded and unliganded PR (43, 62, 79). The serine- threonine protein kinase ck2 (formerly casein kinase II) is ubiquitously expressed with over 300...400, 554, 676 (Zhang et al., 1997; Knotts et al., 2001) and threonine 430 (Knotts et al., 2001). Additionally, while Ser294 is phosphorylated by MAPK

Rheology and phase behavior of dense casein micelle dispersions

NASA Astrophysics Data System (ADS)

Bouchoux, A.; Debbou, B.; Gésan-Guiziou, G.; Famelart, M.-H.; Doublier, J.-L.; Cabane, B.

2009-10-01

Casein micelle dispersions have been concentrated through osmotic stress and examined through rheological experiments. In conditions where the casein micelles are separated from each other, i.e., below random-close packing, the dispersions have exactly the flow and dynamic properties of the polydisperse hard-sphere fluid, demonstrating that the micelles interact only through excluded volume effects in this regime. These interactions cause the viscosity and the elastic modulus to increase by three orders of magnitude approaching the concentration of random-close packing estimated at Cmax≈178 g/l. Above Cmax, the dispersions progressively turn into "gels" (i.e., soft solids) as C increases, with elastic moduli G' that are nearly frequency independent. In this second regime, the micelles deform and/or deswell as C increases, and the resistance to deformation results from the formation of bonds between micelles combined with the intrinsic mechanical resistance of the micelles. The variation in G' with C is then very similar to that observed with concentrated emulsions where the resistance to deformation originates from a set of membranes that separate the droplets. As in the case of emulsions, the G' values at high frequency are also nearly identical to the osmotic pressures required to compress the casein dispersions. The rheology of sodium caseinate dispersions in which the caseins are not structured into micelles is also reported. Such dispersions have the behavior of associative polymer solutions at all the concentrations investigated, further confirming the importance of structure in determining the rheological properties of casein micelle systems.

In Vitro Proliferation and Anti-Apoptosis of the Papain-Generated Casein and Soy Protein Hydrolysates towards Osteoblastic Cells (hFOB1.19).

PubMed

Pan, Xiao-Wen; Zhao, Xin-Huai

2015-06-17

Casein and soy protein were digested by papain to three degrees of hydrolysis (DH) 7.3%-13.3%, to obtain respective six casein and soy protein hydrolysates, aiming to clarify their in vitro proliferation and anti-apoptosis towards a human osteoblastic cell line (hFOB1.19 cells). Six casein and soy protein hydrolysates at five levels (0.01-0.2 mg/mL) mostly showed proliferation as positive 17β-estradiol did, because they conferred the osteoblasts with cell viability of 100%-114% and 104%-123%, respectively. The hydrolysates of higher DH values had stronger proliferation. Casein and soy protein hydrolysates of the highest DH values altered cell cycle progression, and enhanced cell proportion of S-phase from 50.5% to 56.5% and 60.5%. The two also antagonized etoposide- and NaF-induced osteoblast apoptosis. In apoptotic prevention, apoptotic cells were decreased from 31.6% to 22.6% and 15.6% (etoposide treatment), or from 19.5% to 17.7% and 12.4% (NaF treatment), respectively. In apoptotic reversal, soy protein hydrolysate decreased apoptotic cells from 13.3% to 11.7% (etoposide treatment), or from 14.5% to 11.0% (NaF treatment), but casein hydrolysate showed no reversal effect. It is concluded that the hydrolysates of two kinds had estradiol-like action on the osteoblasts, and soy protein hydrolysates had stronger proliferation and anti-apoptosis on the osteoblasts than casein hydrolysates.

Impact of casein and egg white proteins on the structure of wheat gluten-based protein-rich food.

PubMed

Wouters, Arno G B; Rombouts, Ine; Lagrain, Bert; Delcour, Jan A

2016-02-01

There is a growing interest in texturally and nutritionally satisfying vegetable alternatives to meat. Wheat gluten proteins have unique functional properties but a poor nutritional value in comparison to animal proteins. This study investigated the potential of egg white and bovine milk casein with well-balanced amino acid composition to increase the quality of wheat gluten-based protein-rich foods. Heating a wheat gluten (51.4 g)-water (100.0 mL) blend for 120 min at 100 °C increased its firmness less than heating a wheat gluten (33.0 g)-freeze-dried egg white (16.8 g)-water (100.0 mL) blend. In contrast, the addition of casein to the gluten-water blend negatively impacted firmness after heating. Firmness was correlated with loss of protein extractability in sodium dodecyl sulfate containing medium during heating, which was higher with egg white than with casein. Even more, heat-induced polymerization of the gluten-water blend with egg white but not with casein was greater than expected from the losses in extractability of gluten and egg white on their own. Structure formation was favored by mixing gluten with egg white but not with casein. These observations were linked to the intrinsic polymerization behavior of egg white and casein, but also to their interaction with gluten. Thus not all nutritionally suitable proteins can be used for enrichment of gluten-based protein-rich foods. © 2015 Society of Chemical Industry.

Mixed micellization between natural and synthetic block copolymers: β-casein and Lutrol F-127.

PubMed

Portnaya, Irina; Khalfin, Rafail; Kesselman, Ellina; Ramon, Ory; Cogan, Uri; Danino, Dganit

2011-02-28

Amphiphilic block copolymers and mixtures of amphiphiles find broad applications in numerous technologies, including pharma, food, cosmetic and detergency. Here we report on the interactions between a biological charged diblock copolymer, β-casein, and a synthetic uncharged triblock copolymer, Lutrol F-127 (EO(101)PO(56)EO(101)), on their mixed micellization characteristics and the micelles' structure and morphology. Isothermal titration calorimetry (ITC) experiments indicate that mixed micelles form when Lutrol is added to monomeric as well as to assembled β-casein. The main driving force for the mixed micellization is the hydrophobic interactions. Above β-casein CMC, strong perturbations caused by penetration of the hydrophobic oxypropylene sections of Lutrol into the protein micellar core lead to disintegration of the micelles and reformation of mixed Lutrol/β-casein micelles. The negative enthalpy of micelle formation (ΔH) and cooperativity increase with raising β-casein concentration in solution. ζ-potential measurements show that Lutrol interacts with the protein micelles to form mixed micelles even below its critical micellization temperature (CMT). They further indicate that Lutrol effectively masks the protein charges, probably by forming a coating layer of the ethyleneoxide rich chains. Small-angle X-ray scattering (SAXS) and cryogenic-transmission electron microscopy (cryo-TEM) indicate relatively small changes in the oblate micellar shape, but do show swelling along the small axis of β-casein micelles in the presence of Lutrol, thereby confirming the formation of mixed micelles.

Rheology and phase behavior of dense casein micelle dispersions.

PubMed

Bouchoux, A; Debbou, B; Gésan-Guiziou, G; Famelart, M-H; Doublier, J-L; Cabane, B

2009-10-28

Casein micelle dispersions have been concentrated through osmotic stress and examined through rheological experiments. In conditions where the casein micelles are separated from each other, i.e., below random-close packing, the dispersions have exactly the flow and dynamic properties of the polydisperse hard-sphere fluid, demonstrating that the micelles interact only through excluded volume effects in this regime. These interactions cause the viscosity and the elastic modulus to increase by three orders of magnitude approaching the concentration of random-close packing estimated at C(max) approximately 178 g/l. Above C(max), the dispersions progressively turn into "gels" (i.e., soft solids) as C increases, with elastic moduli G(') that are nearly frequency independent. In this second regime, the micelles deform and/or deswell as C increases, and the resistance to deformation results from the formation of bonds between micelles combined with the intrinsic mechanical resistance of the micelles. The variation in G(') with C is then very similar to that observed with concentrated emulsions where the resistance to deformation originates from a set of membranes that separate the droplets. As in the case of emulsions, the G(') values at high frequency are also nearly identical to the osmotic pressures required to compress the casein dispersions. The rheology of sodium caseinate dispersions in which the caseins are not structured into micelles is also reported. Such dispersions have the behavior of associative polymer solutions at all the concentrations investigated, further confirming the importance of structure in determining the rheological properties of casein micelle systems.

The impact of the concentration of casein micelles and whey protein-stabilized fat globules on the rennet-induced gelation of milk.

PubMed

Gaygadzhiev, Zafir; Corredig, Milena; Alexander, Marcela

2009-02-01

The rennet-induced aggregation of skim milk recombined with whey protein-stabilized emulsion droplets was studied using diffusing wave spectroscopy (DSW) and small deformation rheology. The effect of different volume fractions of casein micelles and fat globules was investigated by observing changes in turbidity (1/l*), apparent radius, elastic modulus and mean square displacement (MSD), in addition to confocal imaging of the gels. Skim milk containing different concentration of casein micelles showed comparable light-scattering profiles; a higher volume fraction of caseins led to the development of more elastic gels. By following the development of 1/l* in recombined milks, it was possible to describe the behaviour of the fat globules during the initial stages of rennet coagulation. Increasing the volume fraction of fat globules showed a significant increase in gel elasticity, caused by flocculation of the oil droplets. The presence of flocculated oil globules within the gel structure was confirmed by confocal microscopy observations. Moreover, a lower degree of kappa-casein hydrolysis was needed to initiate casein micelles aggregation in milk containing whey protein-stabilized oil droplets compared to skim milk. This study for the first time clearly describes the impact of a mixture of casein micelles and whey protein-stabilized fat globules on the pre-gelation stages of rennet coagulation, and further highlights the importance of the flocculation state of the emulsion droplets in affecting the structure formation of the gel.

Lactobacillus helveticus as a tool to change proteolysis and functionality in Swiss-type cheeses.

PubMed

Sadat-Mekmene, L; Richoux, R; Aubert-Frogerais, L; Madec, M-N; Corre, C; Piot, M; Jardin, J; le Feunteun, S; Lortal, S; Gagnaire, V

2013-03-01

Lactobacillus helveticus exhibits a great biodiversity in terms of protease gene content, with 1 to 4 cell envelope proteinases. Among them, proteinases PrtH and PrtH2 were shown to have different cleavage specificity on pure α(s1)-casein. The aim of this work was to investigate the proteolytic activity of 2L. helveticus strains in cheese matrix: ITGLH77 (PrtH2 only) and ITGLH1 (at least 2 proteinases, PrtH and PrtH2). Cell viability, proteolysis, autolysis, and stretchability of experimental Emmental cheeses were measured during ripening. The peptides identified by mass spectrometry showed very different profiles in the 2 cheeses. Regardless of the casein origin, the number of different peptides containing more than 20 amino acids was greater in cheeses manufactured with strain ITGLH77. This accumulation of large peptides, including those from α(s1)- and α(s2)-caseins, was in agreement with the lower overall extent of proteolysis obtained in ITGLH77 cheeses, which can be attributed to the presence of one cell envelope proteinase of the lactobacilli strains or lesser release of intracellular peptidases into the cheese aqueous phase. In parallel, stretchability was measured throughout ripening time. Emmental strands observed by confocal laser scanning microscopy showed microstructure similar to that of mozzarella strands. Stretchability was correlated with a specific type of peptide (hydrophobic), as shown by principal component analysis, and with a lower degree of proteolysis. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Purification and characterization of a novel milk-clotting metalloproteinase from Paenibacillus spp. BD3526.

PubMed

Hang, Feng; Wang, Qinbo; Hong, Qing; Liu, Peiyi; Wu, Zhengjun; Liu, Zhenmin; Zhang, Hao; Chen, Wei

2016-04-01

In this study, a milk-clotting enzyme (MCE) isolated from Paenibacillus spp. BD3526 was purified and characterized. The MCE was purified 8.9-fold with a 10.11% recovery using ammonium sulfate precipitation and anion-exchange chromatography and the specific milk-clotting activity (MCA) reached 6791.73 SU/mg. The enzyme was characterized as a 35kDa metalloproteinase, and the zymogen of which was encoded by a 1671 bp gene named zinc metalloproteinase precursor (zmp) with a predicted molecular weight of 59.6 kDa. The optimal temperature for MCA and proteolytic activity (PA) was 65°C and 60°C, respectively. The enzyme was stable over a pH range of 5.0-9.0 and at temperatures below 50°C. The MCA was completely inactivated when the enzyme was heated at 60°C for 30 min, and the PA was totally inactivated for 20 and 10 min when the enzyme was heated at 55°C and 60°C, respectively. The BD3526 enzyme was preferentially active towards κ-casein (κ-CN) and β-casein (β-CN), as determined by sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE), whereas the hydrolysis of αs-casein (αs-CN) was slow and comparable to that caused by chymosin and asparatic acid proteinase from Rhizomucor miehei. The cleavage site of the metalloproteinase in κ-CN was located at the Met106-Ala107 bond, as determined by mass spectrometry analysis. Copyright © 2016. Published by Elsevier B.V.

Discovery of a protein phosphatase activity encoded in the genome of bacteriophage lambda. Probable identity with open reading frame 221.

PubMed

Cohen, P T; Cohen, P

1989-06-15

Infection of Escherichia coli with phage lambda gt10 resulted in the appearance of a protein phosphatase with activity towards 32P-labelled casein. Activity reached a maximum near the point of cell lysis and declined thereafter. The phosphatase was stimulated 30-fold by Mn2+, while Mg2+ and Ca2+ were much less effective. Activity was unaffected by inhibitors 1 and 2, okadaic acid, calmodulin and trifluoperazine, distinguishing it from the major serine/threonine-specific protein phosphatases of eukaryotic cells. The lambda phosphatase was also capable of dephosphorylating other substrates in the presence of Mn2+, although activity towards 32P-labelled phosphorylase was 10-fold lower, and activity towards phosphorylase kinase and glycogen synthase 25 50-fold lower than with casein. No casein phosphatase activity was present in either uninfected cells, or in E. coli infected with phage lambda gt11. Since lambda gt11 lacks part of the open reading frame (orf) 221, previously shown to encode a protein with sequence similarity to protein phosphatase-1 and protein phosphatase-2A of mammalian cells [Cohen, Collins, Coulson, Berndt & da Cruz e Silva (1988) Gene 69, 131-134], the results indicate that ORF221 is the protein phosphatase detected in cells infected with lambda gt10. Comparison of the sequence of ORF221 with other mammalian protein phosphatases defines three highly conserved regions which are likely to be essential for function. The first of these is deleted in lambda gt11.

Effect of solvent and temperature on the size distribution of casein micelles measured by dynamic light scattering.

PubMed

Beliciu, C M; Moraru, C I

2009-05-01

The objectives of this study were to investigate the effect of the solvent on the accuracy of casein micelle particle size determination by dynamic light scattering (DLS) at different temperatures and to establish a clear protocol for these measurements. Dynamic light scattering analyses were performed at 6, 20, and 50 degrees C using a 90Plus Nanoparticle Size Analyzer (Brookhaven Instruments, Holtsville, NY). Raw and pasteurized skim milk were used as sources of casein micelles. Simulated milk ultrafiltrate, ultrafiltered water, and permeate obtained by ultrafiltration of skim milk using a 10-kDa cutoff membrane were used as solvents. The pH, ionic concentration, refractive index, and viscosity of all solvents were determined. The solvents were evaluated by DLS to ensure that they did not have a significant influence on the results of the particle size measurements. Experimental protocols were developed for accurate measurement of particle sizes in all solvents and experimental conditions. All measurements had good reproducibility, with coefficients of variation below 5%. Both the solvent and the temperature had a significant effect on the measured effective diameter of the casein micelles. When ultrafiltered permeate was used as a solvent, the particle size and polydispersity of casein micelles decreased as temperature increased. The effective diameter of casein micelles from raw skim milk diluted with ultrafiltered permeate was 176.4 +/- 5.3 nm at 6 degrees C, 177.4 +/- 1.9 nm at 20 degrees C, and 137.3 +/- 2.7 nm at 50 degrees C. This trend was justified by the increased strength of hydrophobic bonds with increasing temperature. Overall, the results of this study suggest that the most suitable solvent for the DLS analyses of casein micelles was casein-depleted ultrafiltered permeate. Dilution with water led to micelle dissociation, which significantly affected the DLS measurements, especially at 6 and 20 degrees C. Simulated milk ultrafiltrate seemed to give accurate results only at 20 degrees C. Results obtained in simulated milk ultrafiltrate at 6 degrees C could not be explained based on the known effects of temperature on the casein micelle, whereas at 50 degrees C, precipitation of amorphous calcium phosphate affected the DLS measurement.

Characterization of Three Novel Fatty Acid- and Retinoid-Binding Protein Genes (Ha-far-1, Ha-far-2 and Hf-far-1) from the Cereal Cyst Nematodes Heterodera avenae and H. filipjevi.

PubMed

Qiao, Fen; Luo, Lilian; Peng, Huan; Luo, Shujie; Huang, Wenkun; Cui, Jiangkuan; Li, Xin; Kong, Lingan; Jiang, Daohong; Chitwood, David J; Peng, Deliang

2016-01-01

Heterodera avenae and H. filipjevi are major parasites of wheat, reducing production worldwide. Both are sedentary endoparasitic nematodes, and their development and parasitism depend strongly on nutrients obtained from hosts. Secreted fatty acid- and retinol-binding (FAR) proteins are nematode-specific lipid carrier proteins used for nutrient acquisition as well as suppression of plant defenses. In this study, we obtained three novel FAR genes Ha-far-1 (KU877266), Ha-far-2 (KU877267), Hf-far-1 (KU877268). Ha-far-1 and Ha-far-2 were cloned from H. avenae, encoding proteins of 191 and 280 amino acids with molecular masses about 17 and 30 kDa, respectively and sequence identity of 28%. Protein Blast in NCBI revealed that Ha-FAR-1 sequence is 78% similar to the Gp-FAR-1 protein from Globodera pallida, while Ha-FAR-2 is 30% similar to Rs-FAR-1 from Radopholus similis. Only one FAR protein Hf-FAR-1was identified in H. filipjevi; it had 96% sequence identity to Ha-FAR-1. The three proteins are alpha-helix-rich and contain the conserved domain of Gp-FAR-1, but Ha-FAR-2 had a remarkable peptide at the C-terminus which was random-coil-rich. Both Ha-FAR-1 and Hf-FAR-1 had casein kinase II phosphorylation sites, while Ha-FAR-2 had predicted N-glycosylation sites. Phylogenetic analysis showed that the three proteins clustered together, though Ha-FAR-1 and Hf-FAR-1 adjoined each other in a plant-parasitic nematode branch, but Ha-FAR-2 was distinct from the other proteins in the group. Fluorescence-based ligand binding analysis showed the three FAR proteins bound to a fluorescent fatty acid derivative and retinol and with dissociation constants similar to FARs from other species, though Ha-FAR-2 binding ability was weaker than that of the two others. In situ hybridization detected mRNAs of Ha-far-1 and Ha-far-2 in the hypodermis. The qRT-PCR results showed that the Ha-far-1and Ha-far-2 were expressed in all developmental stages; Ha-far-1 expressed 70 times more than Ha-far-2 in all stages. The highest expression level of Ha-far-1 was observed in fourth-stage juvenile (J4), whereas the highest expression level of Ha-far-2 occurred in second-stage juvenile (J2). In conclusion, we have identified two novel far genes from H. avenae and one from H. filipjevi and have provided further indication that nematode far genes are present in a variety of nematode species, where the FAR proteins share similar basic structure, expression pattern and biochemical activities.

Casein phosphopeptides and CaCl2 increase penicillin production and cause an increment in microbody/peroxisome proteins in Penicillium chrysogenum.

PubMed

Domínguez-Santos, Rebeca; Kosalková, Katarina; García-Estrada, Carlos; Barreiro, Carlos; Ibáñez, Ana; Morales, Alejandro; Martín, Juan-Francisco

2017-03-06

Transport of penicillin intermediates and penicillin secretion are still poorly characterized in Penicillium chrysogenum (re-identified as Penicillium rubens). Calcium (Ca 2+ ) plays an important role in the metabolism of filamentous fungi, and casein phosphopeptides (CPP) are involved in Ca 2+ internalization. In this study we observe that the effect of CaCl 2 and CPP is additive and promotes an increase in penicillin production of up to 10-12 fold. Combination of CaCl 2 and CPP greatly promotes expression of the three penicillin biosynthetic genes. Comparative proteomic analysis by 2D-DIGE, identified 39 proteins differentially represented in P. chrysogenum Wisconsin 54-1255 after CPP/CaCl 2 addition. The most interesting group of overrepresented proteins were a peroxisomal catalase, three proteins of the methylcitrate cycle, two aminotransferases and cystationine β-synthase, which are directly or indirectly related to the formation of penicillin amino acid precursors. Importantly, two of the enzymes of the penicillin pathway (isopenicillin N synthase and isopenicillin N acyltransferase) are clearly induced after CPP/CaCl 2 addition. Most of these overrepresented proteins are either authentic peroxisomal proteins or microbody-associated proteins. This evidence suggests that addition of CPP/CaCl 2 promotes the formation of penicillin precursors and the penicillin biosynthetic enzymes in peroxisomes and vesicles, which may be involved in transport and secretion of penicillin. Penicillin biosynthesis in Penicillium chrysogenum is one of the best characterized secondary metabolism processes. However, the mechanism by which penicillin is secreted still remains to be elucidated. Taking into account the role played by Ca 2+ and CPP in the secretory pathway and considering the positive effect that Ca 2+ exerts on penicillin production, the analysis of global protein changes produced after CPP/CaCl 2 addition is very helpful to decipher the processes related to the biosynthesis and secretion of penicillin. Copyright © 2017 Elsevier B.V. All rights reserved.

A 1H-n.m.r. study of casein micelles.

PubMed Central

Griffin, M C; Roberts, G C

1985-01-01

The 1H-n.m.r. spectrum of casein micelles consists of a small number of moderately sharp (linewidth approx. 60 Hz) resonances superimposed on the envelope of very broad lines expected for particles of this size. These sharp lines resemble, in chemical shift and relative intensity, the spectrum of the isolated 'macropeptide' released from the micelles by treatment with chymosin. The sharp lines in the casein micelle spectrum are further sharpened by addition of chymosin and broadened markedly by addition of ethanol. These observations are consistent with the proposal that the 'macropeptide' (the C-terminal 64 residues of K-casein) forms flexible 'hairs' on the surface of the micelles. PMID:3924034

Preparation, characterisation and antioxidant activities of rutin-loaded zein-sodium caseinate nanoparticles.

PubMed

Zhang, Shuangling; Han, Yue

2018-01-01

Novel rutin-loaded zein-sodium caseinate nanoparticles (ZP) with antioxidant activity in aqueous medium were investigated. The results showed that the sodium caseinate concentrations, dosages of rutin and ethanol volume fractions significantly affected the zein nanoparticles' characteristics. Concerning the antioxidant properties, the highest values of rutin loaded ZP obtained using 2, 2-diphenyl-1-picrylhydrazyl scavenging and 2 and 2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) decolourisation assays were 52.7% and 71.2%, respectively, and the total antioxidant capacity was 0.40 nmol g-1. The results suggest that zein-sodium caseinate nanoparticles can be used as a new nano carrier system for rutin or other water insoluble active ingredients.

Preparation, characterisation and antioxidant activities of rutin-loaded zein-sodium caseinate nanoparticles

PubMed Central

Han, Yue

2018-01-01

Novel rutin-loaded zein-sodium caseinate nanoparticles (ZP) with antioxidant activity in aqueous medium were investigated. The results showed that the sodium caseinate concentrations, dosages of rutin and ethanol volume fractions significantly affected the zein nanoparticles’ characteristics. Concerning the antioxidant properties, the highest values of rutin loaded ZP obtained using 2, 2-diphenyl-1-picrylhydrazyl scavenging and 2 and 2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) decolourisation assays were 52.7% and 71.2%, respectively, and the total antioxidant capacity was 0.40 nmol g-1. The results suggest that zein-sodium caseinate nanoparticles can be used as a new nano carrier system for rutin or other water insoluble active ingredients. PMID:29579133

Newly identified mutations at the CSN1S1 gene in Ethiopian goats affect casein content and coagulation properties of their milk.

PubMed

Mestawet, T A; Girma, A; Adnøy, T; Devold, T G; Vegarud, G E

2013-08-01

Very high casein content and good coagulation properties previously observed in some Ethiopian goat breeds led to investigating the αs1-casein (CSN1S1) gene in these breeds. Selected regions of the CSN1S1 gene were sequenced in 115 goats from 5 breeds (2 indigenous: Arsi-Bale and Somali, 1 exotic: Boer, and 2 crossbreeds: Boer × Arsi-Bale and Boer × Somali). The DNA analysis resulted in 35 new mutations: 3 in exons, 3 in the 5' untranslated region (UTR), and 29 in the introns. The mutations in exons that resulted in an amino acid shift were then picked to evaluate their influence on individual casein content (αs1-, αs2-, β-, and κ-CN), micellar size, and coagulation properties in the milk from the 5 goat breeds. A mutation at nucleotide 10657 (exon 10) involved a transversion: CAG→CCG, resulting in an amino acid exchange Gln77→Pro77. This mutation was associated with the indigenous breeds only. Two new mutations, at nucleotide 6072 (exon 4) and 12165 (exon 12), revealed synonymous transitions: GTC→GTT in Val15 and AGA→AGG in Arg100 of the mature protein. Transitions G→A and C→T at nucleotides 1374 and 1866, respectively, occurred in the 5' UTR, whereas the third mutation involved a transversion T→G at nucleotide location 1592. The goats were grouped into homozygote new (CC), homozygote reference (AA), and heterozygote (CA) based on the nucleotide that involved the transversion. The content of αs1-CN (15.32g/kg) in milk samples of goats homozygous (CC) for this newly identified mutation, Gln77→Pro77 was significantly higher than in milks of heterozygous (CA; 9.05g/kg) and reference (AA; 7.61g/kg) genotype animals. The αs2-, β-, and κ-CN contents showed a similar pattern. Milk from goats with a homozygous new mutation had significantly lower micellar size. Milk from both homozygote and heterozygote new-mutation goats had significantly shorter coagulation rate and stronger gel than the reference genotype. Except the transversion, the sequence corresponded to allele A and presumably derived from it. Therefore, this allele is denoted by A3. All goats from the reference genotype (AA) were homozygous for the allele at nucleotide position 1374 and 1866, whereas all mutations in the 5' UTR existed in a heterozygous form in both heterozygous (CA) and the new mutation (CC) genotype. The newly identified mutation (CC) detected in some of the goat breeds is, therefore, important in selection for genetic improvement and high-quality milk for the emerging goat cheese-producing industries. The finding will also benefit farmers raising these goat breeds due to the increased selling price of goats. Further studies should investigate the effect of this amino acid exchange on the secondary and tertiary structure of the αs1-CN molecule and on the susceptibility of peptide hydrolysis by digestive enzymes. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Eya4 Induces Hypertrophy via Regulation of p27kip1.

PubMed

Williams, Tatjana; Hundertmark, Moritz; Nordbeck, Peter; Voll, Sabine; Arias-Loza, Paula Anahi; Oppelt, Daniel; Mühlfelder, Melanie; Schraut, Susanna; Elsner, Ines; Czolbe, Martin; Seidlmayer, Lea; Heinze, Britta; Hahner, Stefanie; Heinze, Katrin; Schönberger, Jost; Jakob, Peter; Ritter, Oliver

2015-12-01

E193, a heterozygous truncating mutation in the human transcription cofactor Eyes absent 4 (Eya4), causes hearing impairment followed by dilative cardiomyopathy. In this study, we first show Eya4 and E193 alter the expression of p27(kip1) in vitro, suggesting Eya4 is a negative regulator of p27. Next, we generated transgenic mice with cardiac-specific overexpression of Eya4 or E193. Luciferase and chromatin immunoprecipitation assays confirmed Eya4 and E193 bind and regulate p27 expression in a contradictory manner. Activity and phosphorylation status of the downstream molecules casein kinase-2α and histone deacetylase 2 were significantly elevated in Eya4- but significantly reduced in E193-overexpressing animals compared with wild-type littermates. Magnetic resonance imaging and hemodynamic analysis indicate Eya4-overexpression results in an age-dependent development of hypertrophy already under baseline conditions with no obvious functional effects, whereas E193 animals develop onset of dilative cardiomyopathy as seen in human E193 patients. Both cardiac phenotypes were aggravated on pressure overload. Finally, we identified a new heterozygous truncating Eya4 mutation, E215, which leads to similar clinical features of disease and a stable myocardial expression of the mutant protein as seen with E193. Our results implicate Eya4/Six1 regulates normal cardiac function via p27/casein kinase-2α/histone deacetylase 2 and indicate that mutations within this transcriptional complex and signaling cascade lead to the development of cardiomyopathy. © 2015 American Heart Association, Inc.

Nano-preparation of Andrographis paniculata extract by casein micelle for antidiabetic agent

NASA Astrophysics Data System (ADS)

Arbianti, Rita; Dewi, Veronica; Imansari, Farisa; Hermansyah, Heri; Sahlan, Muhamad

2017-02-01

Side effects caused by oral medications for person with diabetic are the background of the development of alternative treatments by traditional medicine, herbs. Andrographis paniculata (AP) is one of the herbs that is potent to be anti-diabetic agent. The active compound of AP, andrographolide have been examined to have anti-diabetic activity as α-glucosidase enzyme inhibitor. This research aims to encapsulate sambiloto's extract with casein micelle and produce nanoparticles which have anti-diabetic activity as α-glucosidase inhibitor. Extract of AP is encapsulated by casein micelle and made into nano size using sonicator. The dominant active compounds in AP extract coated by casein are andrographolide, neoandrographolide, 14-deoxy-11,12didehydroandrographolide with encapsulation efficiency of 68.83%, 89.15% and 81.69%, the average diameter of the particles is about 120.57 nm and its loading capacity is 28.85%. AP's extract has antidiabetic activity as α-glucosidase inhibitor with percent inhibition of 95%. The morphology of nanoencapsulated AP's extract analyzed by FE-SEM, were similar with casein micelle.

Phase behavior, rheological characteristics and microstructure of sodium caseinate-Persian gum system.

PubMed

Sadeghi, Farzad; Kadkhodaee, Rassoul; Emadzadeh, Bahareh; Phillips, Glyn O

2018-01-01

In this study, the phase behavior of sodium caseinate-Persian gum mixtures was investigated. The effect of thermodynamic incompatibility on phase distribution of sodium caseinate fractions as well as the flow behavior and microstructure of the biopolymer mixtures were also studied. The phase diagram clearly demonstrated the dominant effect of Persian gum on the incompatibility of the two biopolymers. SDS-PAGE electrophoresis indicated no selective fractionation of sodium caseinate subunits between equilibrium phases upon de-mixing. The microstructure of mixtures significantly changed depending on their position within the phase diagram. Fitting viscometric data to Cross and Bingham models revealed that the apparent viscosity, relaxation time and shear thinning behavior of the mixtures is greatly influenced by the volume ratio and concentration of the equilibrium phases. There is a strong dependence of the flow behavior of sodium caseinate-Persian gum mixtures on the composition of the equilibrium phases and the corresponding microstructure of the system. Copyright © 2017. Published by Elsevier Ltd.

Enhanced pattern resolution, swelling-behaviour and biocompatibility of bioimprinted casein microdevices

NASA Astrophysics Data System (ADS)

Hashemi, Azadeh; de Decker, Fanny; Orcheston-Findlay, Louise; Ali, M. Azam; Alkaisi, Maan M.; Nock, Volker

2017-11-01

This work introduces casein microstructures with surface features as a biodegradable biomedical platform technology for enhancing tissue-engineering applications. An optimized fabrication process is presented to reduce the hydrophobicity of intermediate polydimethylsiloxane (PDMS) molds and to transfer high-resolution regular and biomimetic features onto the surface of casein devices. Four different cross-linking reagents, glutaraldehyde, formaldehyde, citric acid and transglutaminase (TG) were investigated to increase the degradation time of casein and their influence on swelling and biocompatibility of the films was studied. TG was found to be the only cross-linker to effectively increase the degradation time and show reduced film swelling after immersion into media, while remaining compatible with cell-culture. The maximum expansion of the films cross-linked via TG was 33% after 24 hours of immersion in cell-culture media. C2C12 cells were successfully cultured on the patterned films for up to 72 hours. The patterned biodegradable casein substrates presented here have promising applications in stem-cell engineering, regenerative medicine, and implantable devices.

Casein Hydrolysates by Lactobacillus brevis and Lactococcus lactis Proteases: Peptide Profile Discriminates Strain-Dependent Enzyme Specificity.

PubMed

Bounouala, Fatima Zohra; Roudj, Salima; Karam, Nour-Eddine; Recio, Isidra; Miralles, Beatriz

2017-10-25

Casein from ovine and bovine milk were hydrolyzed with two extracellular protease preparations from Lactobacillus brevis and Lactococcus lactis. The hydrolysates were analyzed by HPLC-MS/MS for peptide identification. A strain-dependent peptide profile could be observed, regardless of the casein origin, and the specificity of these two proteases could be computationally ascribed. The cleavage pattern yielding phenylalanine, leucine, or tyrosine at C-terminal appeared both at L. lactis and Lb. brevis hydrolysates. However, the cleavage C-terminal to lysine was favored with Lb. brevis protease. The hydrolysates showed ACE-inhibitory activity with IC 50 in the 16-70 μg/mL range. Ovine casein hydrolysates yielded greater ACE-inhibitory activity. Previously described antihypertensive and opioid peptides were found in these ovine and bovine casein hydrolysates and prediction of the antihypertensive activity of the sequences based on quantitative structure and activity relationship (QSAR) was performed. This approach might represent a useful classification tool regarding health-related properties prior to further purification.

High pressure-assisted encapsulation of vitamin D2 in reassembled casein micelles

NASA Astrophysics Data System (ADS)

Menéndez-Aguirre, O.; Stuetz, W.; Grune, T.; Kessler, A.; Weiss, J.; Hinrichs, J.

2011-03-01

For the encapsulation of vitamin D2, native casein micelles and vitamin D2 with or without additional Ca2+-Pi were treated at 600 MPa and 37 °C for 60 min. The pressure release rate was set at 20 or 600 MPa/min. Vitamin D2 was quantified by reversed-phase high-performance liquid chromatography, and physical properties of the micelles were analysed by photon correlation spectroscopy. The results demonstrate that simultaneous application of Ca2+-Pi and high pressure treatment with a fast release rate significantly increased loading of vitamin D2 per casein by 6.9-fold. The addition of Ca2+-Pi enhanced micelle aggregation and the vitamin was entrapped within the formed aggregates. However, high pressure treatment without Ca2+-Pi with a slow pressure release rate revealed similar results, increasing vitamin D2 per casein by 6.7-fold. The vitamin D2 loading in reassembled casein micelles is supposed to be due to hydrophobic interactions between the hydrophobic domains of the micelles.

Heat-induced gelation of casein micelles in aqueous suspensions at different pH.

PubMed

Thomar, Peggy; Nicolai, Taco

2016-10-01

Heat-induced gelation of casein micelles in aqueous solution was investigated between pH 5.2 and pH 6.7 over a wide range of protein concentrations (C=25-160gL(-1)). For C≥40gL(-1) the casein micelles rapidly formed a self-supporting gel above a critical temperature (Tc). At C=160gL(-1), Tc decreased from 90°C at pH 6.5 to 30°C at pH 5.4 and increased with decreasing protein concentration. Oscillatory shear measurements during heating showed that the elastic modulus (Gel) of the gels increased strongly with increasing protein concentration, but was insensitive to the pH and the heating temperature except close to Tc where Gel decreased sharply with decreasing temperature. The microstructure of the gels was observed by confocal scanning laser microscopy. Heat-induced gelation of casein micelles was compared with that of sodium caseinate solutions free of calcium phosphate. Copyright © 2016 Elsevier B.V. All rights reserved.

Spectroscopic investigation of the influence of calcium ion on the structures of casein micelles.

PubMed

Wang, Peng-Jie; Wu, Jian-Ping; Zhang, Hao; Guo, Hui-Yuan; Liu, Hong-Na; Ren, Fa-Zheng

2014-01-01

The effects of calcium ion on the structural properties of casein micelles in the course of heat treatment were synthetically examined by non-structure-invasive spectrometry. The hydrophobicity, reflected by extrinsic fluorescence (ANS fluorescence), was positively correlated with the concentration of the calcium ion, within the range of 0 to 12 mmol x L(-1). Meanwhile, the turbidity and stability of casein micelles also increased with the growth of calcium concentrations. However, opposite results were observed for hydrodynamic diameter and polydispersity index. Compared with the calcium ion, the calcium-chelator (citrate) has an opposite effect on the structural characteristics of casein micelles. Within the calcium concentrations range of 0 to 12 mmol x L(-1), the hydrophobicity, stability and turbidity were negatively correlated with the concentration of the calcium ion, nevertheless, opposite results were observed for hydrodynamic diameter and polydispersity index. All the results indicate that the calcium ion could be used to modify the structures of casein micelles during heat heatment.

Ultrasound effects on the assembly of casein micelles in reconstituted skim milk.

PubMed

Liu, Zheng; Juliano, Pablo; Williams, Roderick P W; Niere, Julie; Augustin, Mary Ann

2014-05-01

Reconstituted skim milks (10 % w/w total solids, pH 6·7-8·0) were ultrasonicated (20, 400 or 1600 kHz at a specific energy input of 286 kJ/kg) at a bulk milk temperature of <30 °C. Application of ultrasound to milk at different pH altered the assembly of the casein micelle in milk, with greater effects at higher pH and lower frequency. Low frequency ultrasound caused greater disruption of casein micelles causing release of protein from the micellar to the serum phase than high frequency. The released protein re-associated to form aggregates of smaller size but with surface charge similar to the casein micelles in the original milk. Ultrasound may be used as a physical intervention to alter the size of the micelles and the partitioning of caseins between the micellar and serum phases in milk. The altered protein equilibria induced by ultrasound treatment may have potential for the development of milk with novel functionality.

Improvement of ACE inhibitory activity of casein hydrolysate by Maillard reaction with xylose.

PubMed

Hong, Xu; Meng, Jun; Lu, Rong-Rong

2015-01-01

The Maillard reaction is widely used to improve the functional properties or biological activities of food. The purpose of this study was to investigate the effect of the Maillard reaction on angiotensin I converting enzyme (ACE) inhibitory activity in a casein hydrolysate-xylose system. Two-step hydrolysis was used to prepare casein ACE inhibitory peptides. Maillard reaction products (MRPs) were prepared by heating hydrolyzed casein with xylose at pH 8.0, 110 °C for up to 16 h. The results showed that the content of free amino group decreased (P < 0.05); however, browning intensity and absorbance at 294 nm increased because of the Maillard reaction (P < 0.05). The ACE inhibitory activity improved greatly within 2 h (from 63.48% to 90.23%), which was mainly due to carbonyl ammonia condensation reaction in the MRPs. The study shows that the Maillard reaction under appropriate conditions can improve the ACE inhibitory activity of casein hydrolysate effectively. © 2014 Society of Chemical Industry.

Impact of casein gel microstructure on self-diffusion coefficient of molecular probes measured by 1H PFG-NMR.

PubMed

Le Feunteun, Steven; Mariette, François

2007-12-26

The translational dynamics of poly(ethylene glycol) (PEG) polymers with molecular weights (Mw) varying from 6x10(2) to 5x10(5) were investigated by pulsed field gradient NMR in casein suspensions and in gels induced by acidification, enzyme action, and a combination of both. For molecules with Mwor=8000, there was strong dependence of diffusion on PEG size and on the casein network structure as revealed by scanning electron microscopy images. The diffusion coefficients of the two largest PEGs were increased after coagulation by amounts that depended on the internal structure of the gel. In addition, the 527,000 g/mol PEG was found to deviate from Gaussian diffusion behavior to greater or lesser extents according to the casein concentration and the sample microstructure. The results are discussed in terms of network rearrangements.

Analysis of Casein Biopolymers Adsorption to Lignocellulosic Biomass as a Potential Cellulase Stabilizer

PubMed Central

Eckard, Anahita Dehkhoda; Muthukumarappan, Kasiviswanathan; Gibbons, William

2012-01-01

Although lignocellulosic materials have a good potential to substitute current feedstocks used for ethanol production, conversion of these materials to fermentable sugars is still not economical through enzymatic hydrolysis. High cost of cellulase has prompted research to explore techniques that can prevent from enzyme deactivation. Colloidal proteins of casein can form monolayers on hydrophobic surfaces that alleviate the de-activation of protein of interest. Scanning electron microscope (SEM), fourier transform infrared spectroscopy (FT-IR), capillary electrophoresis (CE), and Kjeldahl and BSA protein assays were used to investigate the unknown mechanism of action of induced cellulase activity during hydrolysis of casein-treated biomass. Adsorption of casein to biomass was observed with all of the analytical techniques used and varied depending on the pretreatment techniques of biomass. FT-IR analysis of amides I and II suggested that the substructure of protein from casein or skim milk were deformed at the time of contact with biomass. With no additive, the majority of one of the cellulase mono-component, 97.1 ± 1.1, was adsorbed to CS within 24 h, this adsorption was irreversible and increased by 2% after 72 h. However, biomass treatment with skim-milk and casein reduced the adsorption to 32.9% ± 6.0 and 82.8% ± 6.0, respectively. PMID:23118515

Short communication: Serum composition of milk subjected to re-equilibration by dialysis at different temperatures, after pH adjustments.

PubMed

Zhao, Zhengtao; Corredig, Milena

2016-04-01

The objective of this work was to investigate the properties of casein micelles after pH adjustment and their re-equilibration to the original pH and serum composition. Re-equilibration was carried out by dialyzing against skim milk at 2 different temperatures (4 or 22 °C). Turbidity, the average radius of the casein micelles, and the composition of the soluble phase were measured at different pH values, ranging between 5.5 and 8. Acidification led to the solubilization of colloidal calcium phosphate and decrease of the average radius of the micelles. With re-equilibration, casein dissociation occurred. In milk with pH values greater than 6.0, the average radius was recovered after re-equilibration. At pH values greater than neutral, an increase of the radius of casein micelles and increased dissociation of the casein were found. After re-equilibration, the radius of micelles and soluble protein in the serum decreased. The results were not affected by the temperature of re-equilibration. The changes to the calcium phosphate equilibrium and the dissociation of the micelles will have important consequences to the functionality of casein micelles. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Casein Micelle Dispersions under Osmotic Stress

PubMed Central

Bouchoux, Antoine; Cayemitte, Pierre-Emerson; Jardin, Julien; Gésan-Guiziou, Geneviève; Cabane, Bernard

2009-01-01

Abstract Casein micelles dispersions have been concentrated and equilibrated at different osmotic pressures using equilibrium dialysis. This technique measured an equation of state of the dispersions over a wide range of pressures and concentrations and at different ionic strengths. Three regimes were found. i), A dilute regime in which the osmotic pressure is proportional to the casein concentration. In this regime, the casein micelles are well separated and rarely interact, whereas the osmotic pressure is dominated by the contribution from small residual peptides that are dissolved in the aqueous phase. ii), A transition range that starts when the casein micelles begin to interact through their κ-casein brushes and ends when the micelles are forced to get into contact with each other. At the end of this regime, the dispersions behave as coherent solids that do not fully redisperse when osmotic stress is released. iii), A concentrated regime in which compression removes water from within the micelles, and increases the fraction of micelles that are irreversibly linked to each other. In this regime the osmotic pressure profile is a power law of the residual free volume. It is well described by a simple model that considers the micelle to be made of dense regions separated by a continuous phase. The amount of water in the dense regions matches the usual hydration of proteins. PMID:19167314

Gluten and casein supplementation does not increase symptoms in children with autism spectrum disorder.

PubMed

Pusponegoro, Hardiono D; Ismael, Sofyan; Firmansyah, Agus; Sastroasmoro, Sudigdo; Vandenplas, Yvan

2015-11-01

A gluten- and casein-free diet is often given to children with autism spectrum disorder (ASD). We aimed to determine the effect of gluten and casein supplementation on maladaptive behaviour, gastrointestinal symptom severity and intestinal fatty acids binding protein (I-FABP) excretion in children with ASD. A randomised, controlled, double-blind trial was performed on 74 children with ASD with severe maladaptive behaviour and increased urinary I-FABP. Subjects were randomised to receive gluten-casein or a placebo for seven days. We evaluated maladaptive behaviour before and after supplementation, using I-FABP excretion, the approach withdrawal problem composite subtest of the Pervasive Developmental Disorder Behavior Inventory and the Gastrointestinal Symptom Severity Index. The mean approach withdrawal problem composite score was significantly higher before supplementation than after, both in the placebo and in the gluten-casein group. However, the mean difference was not significant and may have been caused by additional therapy. There was no significant difference in gastrointestinal symptoms and urinary I-FABP excretion. Administrating gluten-casein to children with ASD for one week did not increase maladaptive behaviour, gastrointestinal symptom severity or urinary I-FABP excretion. The effect of prolonged administration or other mechanisms of enterocyte damage in ASD should be explored. ©2015 Foundation Acta Paediatrica. Published by John Wiley & Sons Ltd.

Study on photophysical and aggregation induced emission recognition of 1,8-naphthalimide probe for casein by spectroscopic method

NASA Astrophysics Data System (ADS)

Sun, Yang; Liu, Zhen; Liang, Xuhua; Fan, Jun; Han, Quan

2013-05-01

A novel water-soluble 1,8-naphthalimide derivative 1, bearing two acetic carboxylic groups, exhibited fluorescent turn-on recognition for casein based on the aggregation induced emission (AIE) character. The photophysical properties of 1 consisting of donor and acceptor units were investigated in different solutions. The fluorescence intensity decreased through taking advantage of twisted intramolecular charge transfer (TICT) and self-association emission with increasing solvent polarity. Moreover, the spectral red-shift and intensity quench in protic solvents were caused by the excited-state hydrogen bond strengthening effect. Density Functional Theory (DFT) calculations revealed that 1 exhibited a strong TICT character. The AIE mechanism of 1 with casein was due to 1 docked in the hydrophobic cavity between sub-micelles and bound with Tyr and Trp residues, resulting in the aggregation of 1 on the casein surface and emission enhancement. Based on this, a novel casein assay method was developed. The proposed exhibited a good linear range from 0.1 to 22 μg mL-1, with the detection limit of 2.8 ng mL-1. Satisfactory reproducibility, reversibility and a short response time were realized. This method was applied to the determination of casein in milk powder samples and the results were in good agreement with the result of Biuret method.

Casein micelle dispersions under osmotic stress.

PubMed

Bouchoux, Antoine; Cayemitte, Pierre-Emerson; Jardin, Julien; Gésan-Guiziou, Geneviève; Cabane, Bernard

2009-01-01

Casein micelles dispersions have been concentrated and equilibrated at different osmotic pressures using equilibrium dialysis. This technique measured an equation of state of the dispersions over a wide range of pressures and concentrations and at different ionic strengths. Three regimes were found. i), A dilute regime in which the osmotic pressure is proportional to the casein concentration. In this regime, the casein micelles are well separated and rarely interact, whereas the osmotic pressure is dominated by the contribution from small residual peptides that are dissolved in the aqueous phase. ii), A transition range that starts when the casein micelles begin to interact through their kappa-casein brushes and ends when the micelles are forced to get into contact with each other. At the end of this regime, the dispersions behave as coherent solids that do not fully redisperse when osmotic stress is released. iii), A concentrated regime in which compression removes water from within the micelles, and increases the fraction of micelles that are irreversibly linked to each other. In this regime the osmotic pressure profile is a power law of the residual free volume. It is well described by a simple model that considers the micelle to be made of dense regions separated by a continuous phase. The amount of water in the dense regions matches the usual hydration of proteins.

Effect of ultra-high pressure homogenization on the interaction between bovine casein micelles and ritonavir.

PubMed

Corzo-Martínez, M; Mohan, M; Dunlap, J; Harte, F

2015-03-01

The aim of this work was to develop a milk-based powder formulation appropriate for pediatric delivery of ritonavir (RIT). Ultra-high pressure homogenization (UHPH) at 0.1, 300 and 500 MPa was used to process a dispersion of pasteurized skim milk (SM) and ritonavir. Loading efficiency was determined by RP-HPLC-UV; characterization of RIT:SM systems was carried out by apparent average hydrodynamic diameter and rheological measurements as well as different analytical techniques including Trp fluorescence, UV spectroscopy, DSC, FTIR and SEM; and delivery capacity of casein micelles was determined by in vitro experiments promoting ritonavir release. Ritonavir interacted efficiently with milk proteins, especially, casein micelles, regardless of the processing pressure; however, results suggest that, at 0.1 MPa, ritonavir interacts with caseins at the micellar surface, whilst, at 300 and 500 MPa, ritonavir is integrated to the protein matrix during UHPH treatment. Likewise, in vitro experiments showed that ritonavir release from micellar casein systems is pH dependent; with a high retention of ritonavir during simulated gastric digestion and a rapid delivery under conditions simulating the small intestine environment. Skim milk powder, especially, casein micelles are potentially suitable and efficient carrier systems to develop novel milk-based and low-ethanol powder formulations of ritonavir appropriate for pediatric applications.

Direct measurements of interfacial interactions between pectin and kappa-casein and implications for the stabilisation of calcium-free casein micelle mimics.

PubMed

Cucheval, A; Al-Ghobashy, M A; Hemar, Y; Otter, D; Williams, M A K

2009-10-15

Using Surface Plasmon Resonance (SPR) it has been shown that the fine structure of the anionic polysaccharide pectin strongly influences its interfacial interaction with a kappa-casein layer coated onto a gold surface (via a dextran linker) in the pH range 3.5-6.8, with the highest SPR signal being observed for pectin with the lowest charge density tested (a degree of methylesterification (DM) around 90%). Furthermore, the Brownian motions of kappa-casein coated polystyrene beads (used to provide calcium-free 'model casein micelles') were studied in pectin solutions using Diffusing Wave Spectroscopy (DWS) and microscopy, and were compared with measurements made on naked beads. At every pH value studied (with the exception of 3.5), bridging of the protein-covered probe particles was observed for pectins of both DM 28 and DM 78. However, no aggregated complexes were found in these model casein micelle systems when pectin of an unusually high DM was used (90%). It was hypothesised that having a limited number of binding regions of spatially limited extent maximises the number of chains binding to the protein layer (as found with the SPR measurement), encourages the formation of loops and trains, and additionally limits the potential for destabilisation via bridging.

Analysis of casein biopolymers adsorption to lignocellulosic biomass as a potential cellulase stabilizer.

PubMed

Eckard, Anahita Dehkhoda; Muthukumarappan, Kasiviswanathan; Gibbons, William

2012-01-01

Although lignocellulosic materials have a good potential to substitute current feedstocks used for ethanol production, conversion of these materials to fermentable sugars is still not economical through enzymatic hydrolysis. High cost of cellulase has prompted research to explore techniques that can prevent from enzyme deactivation. Colloidal proteins of casein can form monolayers on hydrophobic surfaces that alleviate the de-activation of protein of interest. Scanning electron microscope (SEM), fourier transform infrared spectroscopy (FT-IR), capillary electrophoresis (CE), and Kjeldahl and BSA protein assays were used to investigate the unknown mechanism of action of induced cellulase activity during hydrolysis of casein-treated biomass. Adsorption of casein to biomass was observed with all of the analytical techniques used and varied depending on the pretreatment techniques of biomass. FT-IR analysis of amides I and II suggested that the substructure of protein from casein or skim milk were deformed at the time of contact with biomass. With no additive, the majority of one of the cellulase mono-component, 97.1 ± 1.1, was adsorbed to CS within 24 h, this adsorption was irreversible and increased by 2% after 72 h. However, biomass treatment with skim-milk and casein reduced the adsorption to 32.9% ± 6.0 and 82.8% ± 6.0, respectively.

[Allergy due to probable contamination of bread used for school meals with milk].

PubMed

Kiyota, Kyohei; Fujiwara, Yuka; Adachi, Kazuto; Kameda, Makoto; Akutsu, Kazuhiko; Kajimura, Keiji

2014-06-01

In May 2012, two schoolchildren developed allergic symptoms after eating a school meal of fried bread in Osaka, Japan. One specific raw material, milk, should not be present in fried bread. However, we suspected the unintentional mixing of milk in the bread manufacturing process. Our aim was to verify the reason if this was so. We first manufactured bread that contained milk as one of its components. We then thoroughly cleaned the manufacturing line except for the dough divider and, as per the revised instruction manual, continuously manufactured bread that did not contain milk. In this manner, we tried to simulate the conditions at the time of the incident. Casein is a major milk protein. We, therefore, determined casein concentration in the milk-free bread, using casein enzyme-linked immunosorbent assay. We observed casein levels higher than 1000 ppm in the initial lots of breads, but lower levels in the later lots. Casein levels also decreased upon frying of bread. High casein levels, which were observed in the earlier lots of breads, were lower in the later lots. In the manufacturing line, the remnants of the bread dough that contained milk presumably got mixed with the dough used in the subsequent manufacture of milk-free bread.

Mapping a quantitative trait locus for the concentration of beta-lactoglobulin in milk, and the effect of beta-lactoglobulin genetic variants on the composition of milk from Holstein-Friesian x Jersey crossbred cows.

PubMed

Berry, S D; Lopez-Villalobos, N; Beattie, E M; Davis, S R; Adams, L F; Thomas, N L; Ankersmit-Udy, A E; Stanfield, A M; Lehnert, K; Ward, H E; Arias, J A; Spelman, R J; Snell, R G

2010-02-01

To identify quantitative trait loci (QTL) affecting the concentration of beta-lactoglobulin in milk, and to evaluate the effect of beta-lactoglobulin genetic variants on the concentration of fat, protein and casein in bovine milk. A herd of 850 F2 Holstein-Friesian x Jersey crossbred cows was produced through mating six Holstein-Friesian x Jersey F1 bulls of high genetic merit with F1 cows from the national herd. A total of 1,610 herd-test records from 556 second-parity crossbreds were analysed. The concentration of fat, protein and casein in milk was measured at peak, mid- and late lactation, during the production seasons of 2003-2004 and 2004-2005. Liveweight was measured daily. DNA from the F2 animals, their F1 dams and sires, and selected grandsires was genotyped across the genome, initially with 285 microsatellite markers, and subsequently with 6,634 single nucleotide polymorphisms (SNP). A highly significant QTL for the concentration of beta-lactoglobulin in milk was identified, which coincided with the position of the beta-lactoglobulin gene on bovine Chromosome 11. No other consistently significant QTL for the concentration of beta-lactoglobulin in milk were detected. Cows with the BB beta-lactoglobulin genotype produced milk with a 30% lower concentration of beta-lactoglobulin than cows with the AA genotype. The beta-lactoglobulin polymorphism also explained variation in the proportion of casein in total protein. In addition, the percentage of fat was higher for BB than AA animals, whereas the percentage of total protein, mean daily milk yield and liveweight did not differ between AA and BB animals. A significant QTL determining the concentration of beta-lactoglobulin in milk was identified. Selection of animals for the beta-lactoglobulin B-allele may enable the production of milk naturally enriched for casein, thus allowing a potential increase in the yield of cheese. There may be additional future value in production of bovine milk more like human milk, where decreasing the concentration of beta-lactoglobulin is desirable.

Vitamin Requirements of Several Cellulolytic Rumen Bacteria1

PubMed Central

Scott, H. W.; Dehority, B. A.

1965-01-01

Scott, H. W. (Ohio Agricultural Experiment Station, Wooster), and B. A. Dehority. Vitamin requirements of several cellulolytic rumen bacteria. J. Bacteriol. 89:1169–1175. 1965.—Four strains of cellulolytic bacteria recently isolated from in vitro rumen fermentations were used in this study. Nine water-soluble vitamins were tested in single-deletion and single-addition plus biotin experiments, each with and without charcoal-extracted casein hydrolysate. Bacteroides succinogenes A3C and B21a required only biotin under the above experimental conditions. Ruminococcus flavefaciens B34b showed an absolute requirement for biotin and was stimulated by p-aminobenzoic acid (PABA) in the single-deletion experiments. In the single-addition plus biotin experiments, PABA and, to a lesser extent, vitamin B12 appeared to be required for maximal growth. The presence or absence of casein hydrolysate did not affect the vitamin requirements for the aforementioned three strains. In the single-deletion experiments, R. flavefaciens Cla showed an absolute requirement for biotin and, when casein hydrolysate was omitted, for B12. When casein hydrolysate was present, no requirement for B12 could be observed. In the single-addition experiments where the basal medium contained biotin and casein hydrolysate or B12, PABA was required for maximal growth; however, the single deletion of PABA caused only slight retardation of growth. Investigation of the B12 or casein hydrolysate requirement of Cla revealed that a mixture of purified amino acids simulating casein hydrolysate satisfied this requirement. Subsequent work indicated that this requirement could be satisfied by the amino acid methionine. PMID:14292981

Whey or Casein Hydrolysate with Carbohydrate for Metabolism and Performance in Cycling.

PubMed

Oosthuyse, T; Carstens, M; Millen, A M E

2015-07-01

The protein type most suitable for ingestion during endurance exercise is undefined. This study compared co-ingestion of either 15 g/h whey or casein hydrolysate with 63 g/h fructose: maltodextrin (0.8:1) on exogenous carbohydrate oxidation, exercise metabolism and performance. 2 h postprandial, 8 male cyclists ingested either: carbohydrate-only, carbohydrate-whey hydrolysate, carbohydrate-casein hydrolysate or placebo-water in a crossover, double-blind design during 2 h of exercise at 60%W max followed by a 16-km time trial. Data were evaluated by magnitude-based inferential statistics. Exogenous carbohydrate oxidation, measured from (13)CO2 breath enrichment, was not substantially influenced by co-ingestion of either protein hydrolysate. However, only co-ingestion of carbohydrate-casein hydrolysate substantially decreased (98% very likely decrease) total carbohydrate oxidation (mean±SD, 242±44; 258±47; 277±33 g for carbohydrate-casein, carbohydrate-whey and carbohydrate-only, respectively) and substantially increased (93% likely increase) total fat oxidation (92±14; 83±27; 73±19 g) compared with carbohydrate-only. Furthermore, only carbohydrate-casein hydrolysate ingestion resulted in a faster time trial (-3.6%; 90% CI: ±3.2%) compared with placebo-water (95% likely benefit). However, neither protein hydrolysate enhanced time trial performance when compared with carbohydrate-only. Under the conditions of this study, ingesting carbohydrate-casein, but not carbohydrate-whey hydrolysate, favourably alters metabolism during prolonged moderate-strenuous cycling without substantially altering cycling performance compared with carbohydrate-only. © Georg Thieme Verlag KG Stuttgart · New York.

Dietary protein deficiency affects n-3 and n-6 polyunsaturated fatty acids hepatic storage and very low density lipoprotein transport in rats on different diets.

PubMed

Bouziane, M; Prost, J; Belleville, J

1994-04-01

Fatty livers and the similarity between the skin lesions in kwashiorkor and those described in experimental essential fatty acid (EFA) deficiency have led to the hypothesis that protein and EFA deficiencies may both occur in chronic malnutrition. The relationship between serum very low density lipoprotein (VLDL) and hepatic lipid composition was studied after 28 d of protein depletion to determine the interactions between dietary protein levels and EFA availability. Rats were fed purified diets containing 20 or 2% casein and 5% fat as either soybean oil rich in EFA, or salmon oil rich in eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids, or hydrogenated coconut oil poor in EFA. Animals were divided into six groups, SOC (20% casein + 5% soybean oil), SOd (2% casein + 5% soybean oil), COC (20% casein + 5% hydrogenated coconut oil), COd (2% casein + 5% hydrogenated coconut oil), SAC (20% casein + 5% salmon oil) and SAd (2% casein + 5% salmon oil). After 28 d, liver steatosis and reduced VLDL-phospholipid contents (P < 0.001) were observed in protein-deficient rats. In protein deficiency, triacylglycerol and phospholipid fatty acid compositions in both liver and VLDL showed a decreased polyunsaturated-to-saturated fatty acid ratio. This ratio was higher with the salmon oil diets and lower with the hydrogenated coconut oil diets. Furthermore, independent of the oil in the diet, protein deficiency decreased linoleic and arachidonic acids in VLDL phospholipids. Conversely, despite decreased proportions of EPA at low protein levels, DHA levels remained higher in rats fed salmon oil diets.(ABSTRACT TRUNCATED AT 250 WORDS)

Dietary egg-white protein increases body protein mass and reduces body fat mass through an acceleration of hepatic β-oxidation in rats.

PubMed

Matsuoka, Ryosuke; Shirouchi, Bungo; Umegatani, Minami; Fukuda, Meguri; Muto, Ayano; Masuda, Yasunobu; Kunou, Masaaki; Sato, Masao

2017-09-01

Egg-white protein (EWP) is known to reduce lymphatic TAG transport in rats. In this study, we investigated the effects of dietary EWP on body fat mass. Male rats, 4 weeks old, were fed diets containing either 20 % EWP or casein for 28 d. Carcass protein levels and gastrocnemius leg muscle weights in the EWP group were significantly higher than those in the casein group. In addition, carcass TAG levels and abdominal fat weights in the EWP group were significantly lower than those in the casein group; adipocyte size in abdominal fat in the EWP group was smaller than that in the casein group. To identify the involvement of dietary fat levels in the rats, one of two fat levels (5 or 10 %) was added to their diet along with the different protein sources (EWP and casein). Abdominal fat weight and serum and hepatic TAG levels were significantly lower in the EWP group than in the casein group. Moreover, significantly higher values of enzymatic activity related to β-oxidation in the liver were observed in the EWP group compared with the casein group. Finally, abdominal fat weight reduction in the EWP group with the 10 % fat diet was lower than that in the EWP group with the 5 % fat diet. In conclusion, our results indicate that, in addition to the inhibition of dietary TAG absorption reported previously, dietary EWP reduces body fat mass in rats through an increase of body protein mass and the acceleration of β-oxidation in the liver.

Complexation of sodium caseinate with gum tragacanth: Effect of various species and rheology of coacervates.

PubMed

Ghorbani Gorji, Sara; Ghorbani Gorji, Elham; Mohammadifar, Mohammad Amin; Zargaraan, Azizollaah

2014-06-01

We investigated complex coacervation of sodium caseinate/Astragalus rahensis (A.r) as a function of pH with light scattering, spectrophotometry, and viscosity measurements. Interestingly, sodium caseinate/A.r displayed five structural transitions; pH 7.00 to pH ∼5.40: no interaction occurred, pH ∼5.40 to pH ∼4.80: initiation of the formation of primary soluble complexes, pH ∼4.80 to ∼4.30: formation of interpolymer complexes, pH ∼4.30 to ∼4.02: optimum coacervation and pH ∼4.02 to ∼2.50: suppression of coacervation. In addition, rheological properties of sodium caseinate/A.r coacervates were studied at various pH values. A much higher storage modulus (G') than loss modulus (G″) for all sodium caseinate/A.r coacervates suggests the formation of highly interconnected gel-like network structures with mainly elastic behaviour. Moreover, sodium caseinate/A.r coacervates at all pH values exhibited a shear thinning behaviour across the entire shear rate range investigated. Effects of different species of gum tragacanth on the interactions with sodium caseinate have been scarcely studied. Our study showed that systems containing various species (A.r, soluble fraction of A.r and Astragalus gossypinus (A.g)) had different critical pH values and particle sizes during complex coacervation, which could be due to different ratio of soluble to insoluble fractions and uronic acid content of various species. Copyright © 2014 Elsevier B.V. All rights reserved.

[New isolation methods and phylogenetic diversity of actinobacteria from hypersaline beach in Aksu].

PubMed

Zhang, Yao; Xia, Zhanfeng; Cao, Xinbo; Li, Jun; Zhang, Lili

2013-08-04

We explored 4 new methods to improve the isolation of actinobacterial resources from high salt areas. Optimized media based on 4 new strategies were used for isolating actinobacteria from hypersaline beaches. Glycerin-arginine, trehalose-creatine, glycerol-asparticacid, mannitol-casein, casein-mannitol, mannitol-alanine, chitosan-asparagineand GAUZE' No. 1 were used as basic media. New isolation strategy includes 4 methods: ten-fold dilution culture, simulation of the original environment, actinobacterial culture guided by uncultured molecular technology detected, and reference of actinobacterial media for brackish marine environment. The 16S rRNA genes of the isolates were amplified with bacterial universal primers. The results of 16S rRNA gene sequences were compared with sequences obtained from GenBank databases. We constructed phylogenetic tree with the neighbor-joining method. No actinobacterial strains were isolated by 8 media of control group, while 403 strains were isolated by new strategies. The isolates by new methods were members of 14 genera (Streptomyces, Streptomonospora, Saccharomonospora, Plantactinospora, Nocardia, Amycolatopsis, Glycomyces, Micromonospora, Nocardiopsis, Isoptericola, Nonomuraea, Thermobifida, Actinopolyspora, Actinomadura) of 10 families in 8 suborders. The most abundant and diverse isolates were the two suborders of Streptomycineae (69.96%) and Streptosporangineaesuborder (9.68%) within the phylum Actinobacteria, including 9 potential novel species. New isolation methods significantly improved the actinobacterial culturability of hypersaline areas, and obtained many potential novel species, which provided a new and more effective way to isolate actinobacteria resources in hypersaline environments.

Dietary protein level and origin (casein and highly purified soybean protein) affect hepatic storage, plasma lipid transport, and antioxidative defense status in the rat.

PubMed

Madani, S; Prost, J; Belleville, J

2000-05-01

The effects of different proportions (10, 20, and 30%) of dietary casein or highly purified soybean protein on lipid metabolism were studied in growing Wistar rats. Hepatic, plasma and lipoprotein lipid, and protein concentrations, plasma thiobarbituric acid-reactive substance (TBARS) levels, and resistance of red blood cells against free-radical attack were determined after a 4-wk dietary regimen. Compared with the 20% casein diet, the 20% soybean protein diet exhibited similar cholesterolemia but lower plasma triacylglycerol concentrations and very-low-density lipoprotein (VLDL) particle number, as measured by diminished contents of VLDL-triacylglycerol, VLDL-protein, and VLDL-apolipoprotein (Apo) B (B-100 and B-48). The soybean protein diet raised high-density lipoprotein (HDL)(2-3) particle number, as measured by enhanced concentrations of HDL(2-3) cholesterol, HDL-phospholipid, and HDL-ApoA-I. Increasing casein or soybean protein level (from 10 to 30%) in the diet involved higher VLDL-ApoB (B-100 and B-48), indicating an increase in the number of VLDL particles. Feeding the 30% casein or 30% soybean protein diet enhanced LDL-HDL(1) cholesterol contents. Despite similar HDL(2-3)-ApoA-I levels, the 30% casein diet enhanced the HDL(2-3) mass and its cholesterol concentrations. In contrast, feeding either the 10 or 30% soybean protein diet significantly lowered HDL(2-3) cholesterol and ApoA-I levels. These effects on cholesterol distribution in lipoprotein fractions occurred despite unchanged total cholesterol concentrations in plasma. Feeding 20% soybean protein versus 20% casein involved lower plasma TBARS concentrations. Decreasing casein or soybean protein levels in the diet were associated with higher plasma TBARS concentrations and had a lower resistance of red blood cells against free-radical attack. The present study shows that dietary protein level and origin play an important role in lipoprotein metabolism and the antioxidative defense status but do not affect total cholesterol concentrations in plasma.

Polylactic acid composites incorporating casein functionalized cellulose nanowhiskers

PubMed Central

2013-01-01

Background Polylactic acid (PLA) is considered to be a sustainable alternative to petroleum-based polymers for many applications. Using cellulose fiber to reinforce PLA is of great interest recently due to its complete biodegradability and potential improvement of the mechanical performance. However, the dispersion of hydrophilic cellulose fibers in the hydrophobic polymer matrix is usually poor without using hazardous surfactants. The goal of this study was to develop homogenously dispersed cellulose nanowhisker (CNW) reinforced PLA composites using whole milk casein protein, which is an environmentally compatible dispersant. Results In this study, whole milk casein was chosen as a dispersant in the PLA-CNW system because of its potential to interact with the PLA matrix and cellulose. The affinity of casein to PLA was studied by surface plasmon resonance (SPR) imaging. CNWs were functionalized with casein and used as reinforcements to make PLA composites. Fluorescent staining of CNWs in the PLA matrix was implemented as a novel and simple way to analyze the dispersion of the reinforcements. The dispersion of CNWs in PLA was improved when casein was present. The mechanical properties of the composites were studied experimentally. Compared to pure PLA, the PLA composites had higher Young’s modulus. Casein (CS) functionalized CNW reinforced PLA (PLA-CS-CNW) at 2 wt% filler content maintained higher strain at break compared to normal CNW reinforced PLA (PLA-CNW). The Young’s modulus of PLA-CS-CNW composites was also higher than that of PLA-CNW composites at higher filler content. However, all composites exhibited lower strain at break and tensile strength at high filler content. Conclusions The presence of whole milk casein improved the dispersion of CNWs in the PLA matrix. The improved dispersion of CNWs provided higher modulus of the PLA composites at higher reinforcement loading and maintained the strain and stress at break of the composites at relatively low reinforcement loading. The affinity of the dispersant to PLA is important for the ultimate strength and stiffness of the composites. PMID:24341897

Novel sensitive monoclonal antibody based competitive enzyme-linked immunosorbent assay for the detection of raw and processed bovine beta-casein

PubMed Central

Castillo, Daniela S.

2017-01-01

Cow milk protein allergy (CMPA) is the most common childhood food allergy, which can sometimes persist or can newly develop in adulthood with severe symptoms. CMPA's treatment is complete dietary avoidance of milk proteins. To achieve this task, patients have to be aware of milk proteins found as "hidden allergens" in food commodities. In regard to milk proteins, it has been reported that allergenicity of caseins remains unaffected upon heat treatment. For these reasons, we aimed to obtain monoclonal antibodies (mAbs) against native and denatured β-casein, one of the most abundant and antigenic caseins, in order to develop an indirect competitive ELISA (icELISA) to detect and quantify traces of this milk allergen in raw and processed foodstuffs. We developed two specific hybridoma clones, 1H3 and 6A12, which recognized β-casein in its denatured and native conformations by indirect ELISA (iELISA). Cross-reaction analysis by Western blot and iELISA indicated that these mAbs specifically recognized β-casein from bovine and goat milk extracts, while they did not cross-react with proteins present in other food matrixes. These highly specific mAbs enabled the development of sensitive, reliable and reproducible icELISAs to detect and quantify this milk protein allergen in food commodities. The extraction of β-casein from foodstuff was efficiently carried out at 60°C for 15 minutes, using an extraction buffer containing 1% SDS. The present study establishes a valid 1H3 based-icELISA, which allows the detection and quantification -0.29 ppm and 0.80 ppm, respectively- of small amounts of β-casein in raw and processed foods. Furthermore, we were able to detect milk contamination in incurred food samples with the same sensitivity as a commercial sandwich ELISA thus showing that this icELISA constitutes a reliable analytical method for control strategies in food industry and allergy prevention. PMID:28759641

Novel sensitive monoclonal antibody based competitive enzyme-linked immunosorbent assay for the detection of raw and processed bovine beta-casein.

PubMed

Castillo, Daniela S; Cassola, Alejandro

2017-01-01

Cow milk protein allergy (CMPA) is the most common childhood food allergy, which can sometimes persist or can newly develop in adulthood with severe symptoms. CMPA's treatment is complete dietary avoidance of milk proteins. To achieve this task, patients have to be aware of milk proteins found as "hidden allergens" in food commodities. In regard to milk proteins, it has been reported that allergenicity of caseins remains unaffected upon heat treatment. For these reasons, we aimed to obtain monoclonal antibodies (mAbs) against native and denatured β-casein, one of the most abundant and antigenic caseins, in order to develop an indirect competitive ELISA (icELISA) to detect and quantify traces of this milk allergen in raw and processed foodstuffs. We developed two specific hybridoma clones, 1H3 and 6A12, which recognized β-casein in its denatured and native conformations by indirect ELISA (iELISA). Cross-reaction analysis by Western blot and iELISA indicated that these mAbs specifically recognized β-casein from bovine and goat milk extracts, while they did not cross-react with proteins present in other food matrixes. These highly specific mAbs enabled the development of sensitive, reliable and reproducible icELISAs to detect and quantify this milk protein allergen in food commodities. The extraction of β-casein from foodstuff was efficiently carried out at 60°C for 15 minutes, using an extraction buffer containing 1% SDS. The present study establishes a valid 1H3 based-icELISA, which allows the detection and quantification -0.29 ppm and 0.80 ppm, respectively- of small amounts of β-casein in raw and processed foods. Furthermore, we were able to detect milk contamination in incurred food samples with the same sensitivity as a commercial sandwich ELISA thus showing that this icELISA constitutes a reliable analytical method for control strategies in food industry and allergy prevention.

Casein Fermentate of Lactobacillus animalis DPC6134 Contains a Range of Novel Propeptide Angiotensin-Converting Enzyme Inhibitors▿

PubMed Central

Hayes, M.; Stanton, C.; Slattery, H.; O'Sullivan, O.; Hill, C.; Fitzgerald, G. F.; Ross, R. P.

2007-01-01

This work evaluated the angiotensin-converting-enzyme (ACE)-inhibitory activities of a bovine sodium caseinate fermentate generated using the proteolytic capabilities of the porcine small intestinal isolate Lactobacillus animalis DPC6134 (NCIMB deposit 41355). The crude 10-kDa L. animalis DPC6134 fermentate exhibited ACE-inhibitory activity of 85.51% (±15%) and had a 50% inhibitory concentration (IC50) of 0.8 mg protein/ml compared to captopril, which had an IC50 value of 0.005 mg/ml. Fractionation of the crude L. animalis DPC6134 fermentate by membrane filtration and reversed-phase high-performance liquid chromatography (HPLC) generated three bioactive fractions from a total of 72 fractions. Fractions 10, 19, and 43 displayed ACE-inhibitory activity percentages of 67.53 (±15), 83.71 (±19), and 42.36 (±11), respectively, where ACE inhibition was determined with 80 μl of the fractions with protein concentrations of 0.5 mg/ml. HPLC and mass spectrometry analysis identified 25 distinct peptide sequences derived from α-, β-, and κ-caseins. In silico predictions, based on the C-terminal tetrapeptide sequences, suggested that peptide NIPPLTQTPVVVPPFIQ, corresponding to β-casein f(73-89); peptide IGSENSEKTTMP, corresponding to αs1-casein f(201212); peptide SQSKVLPVPQ, corresponding to β-casein f(166-175); peptide MPFPKYPVEP, corresponding to β-casein f(124133); and peptide EPVLGPVRGPFP, corresponding to β-casein f(210-221), contained ACE-inhibitory activities. These peptides were chosen for chemical synthesis to confirm the ACE-inhibitory activity of the fractions. Chemically synthesized peptides displayed IC50 values in the range of 92 μM to 790 μM. Additionally, a simulated gastrointestinal digestion confirmed that the ACE-inhibitory 10-kDa L. animalis DPC6134 fermentation was resistant to a cocktail of digestive enzymes found in the gastrointestinal tract. PMID:17483275