OGR1 negatively regulates ß-casein and triglyceride synthesis and cell proliferation via the PI3K/AKT/mTOR signaling pathway in goat mammary epithelial cells.

Goat milk in some cases is less allergenic than cow milk, therefore, more people drink goat milk in the world, so it is necessary for us to improve the yield and quality of goat milk. Previous studies have shown that some genes are closely related to lactation. Ovarian cancer G protein-coupled 1 (OGR1) is a G protein-coupled receptor discovered recently. OGR1 is widely found in various tissues of organisms and is involved in cell skeleton reorganization, carcinogenesis, cell proliferation, and apoptosis by regulating multiple signaling pathways in cells. However, the modulating effect of OGR1 in lactation is still unknown. Therefore, the objective of this study is to investigate the function of OGR1 in goat mammary epithelial cells (GMECs). Flow cytometry, CCK8, EDU, enzyme-linked immunosorbent assay, and triglyceride test kit assays were performed and we found that OGR1 regulated Bcl-2/Bax ratio, Fas protein expression as well as the phosphorylation of AKT and mammalian target of rapamycin (mTOR). si-OGR1 could enhance the proliferation of GMECs by promoting G1/S phase progression and the synthesis of ß-casein and triglyceride. By contrast, OGR1 repressed GMECs proliferation and down-regulated the synthesis of ß-casein and triglyceride by blocking the PI3K/AKT/mTOR signaling pathway in GMECs.

MiR-204-5p promotes lipid synthesis in mammary epithelial cells by targeting SIRT1.

OBJECTIVES: Understanding the molecular mechanisms of lipid synthesis in the mammary gland is crucial for regulating the level and composition of lipids in milk. This study aimed to investigate the functional and molecular mechanisms of miR-204-5p in mammary epithelial cells to provide a theoretical basis for milk lipid synthesis. METHODS: Real-time quantitative PCR was performed to detect the transcriptional levels of miR-204-5p and related mRNA abundance in mammary epithelial cells. Western blotting was conducted to determine protein expression. Cell proliferation was assessed by Cell Counting Kit-8. A dual-luciferase reporter assay was conducted to verify the targeting relationship between miR-204-5p and SIRT1. siRNA and overexpression plasmids were transfected into mouse HC11 mammary epithelial cells. RESULTS: The abundance of miR-204-5p was much higher in lactating mouse mammary glands than in other tissues, which indicated that miR-204-5p may be involved in regulating milk production. MiR-204-5p affected the expression of ß-casein and milk lipid synthesis in HC11 mouse mammary epithelial cells but did not influence the proliferation of HC11 cells. Overexpression of miR-204-5p significantly increased the number of Oil Red O+ cells, triglyceride accumulation and the expression of markers associated with lipid synthesis, including FASN and PPARγ, whereas inhibition of miR-204-5p had the opposite effect. miR-204-5p promotes lipid synthesis by negatively regulating SIRT1. Overexpression of SIRT1 can repress the promotion of miR-204-5p on lipid synthesis. CONCLUSION: Our findings showed that miR-204-5p can promote the synthesis of milk lipids in mammary epithelial cells by targeting SIRT1.

High levels of fatty acids inhibit ß-casein synthesis through suppression of the JAK2/STAT5 and mTOR signaling pathways in mammary epithelial cells of cows with clinical ketosis.

Ketosis is a metabolic disease of dairy cows often characterized by high concentrations of ketone bodies and fatty acids, but low milk protein and milk production. The Janus kinase 2 (JAK2)-signal transducer and activator of transcription 5 (STAT5) and the mechanistic target of rapamycin (mTOR) signaling pathways are central for the regulation of milk protein synthesis. The effect of high levels of fatty acids on these pathways and ß-casein synthesis are unknown in dairy cows with clinical ketosis. Mammary gland tissue and blood samples were collected from healthy (n = 15) and clinically-ketotic (n = 15) cows. In addition, bovine mammary epithelial cells (BMEC) were treated with fatty acids, methionine (Met) or prolactin (PRL), respectively. In vivo, the serum concentration of fatty acids was greater (P > 0.05) and the percentage of milk protein (P > 0.05) was lower in cows with clinical ketosis. The JAK2-STAT5 and mTOR signaling pathways were inhibited and the abundance of ß-casein was lower in mammary tissue of cows with clinical ketosis (P > 0.05). In vitro, high levels of fatty acids inhibited the JAK2-STAT5 and mTOR signaling pathways (P > 0.05) and further decreased the ß-casein synthesis (P > 0.05) in BMEC. Methionine or PRL treatment, as positive regulators, activated the JAK2-STAT5 and mTOR signaling pathways to increase the ß-casein synthesis. Importantly, the high concentration of fatty acids attenuated the positive effect of Met or PRL on mTOR, JAK2-STAT5 pathways and the abundance of ß-casein (P > 0.05). Overall, these data indicate that the high concentrations of fatty acids that reach the mammary cells during clinical ketosis inhibit mTOR and JAK2-STAT5 signaling pathways, and further suppress ß-casein synthesis.

ASCT2 Is Involved in SARS-Mediated ß-Casein Synthesis of Bovine Mammary Epithelial Cells with Methionine Supply.

The methionine (Met) uptake into mammary cells depends upon the corresponding amino acid (AA) transporters, which play a regulatory role in the mammary protein production beyond transport. Our previous studies have identified that seryl-tRNA synthetase (SARS) could be a novel mediator to regulate essential AA-stimulated casein synthesis in primary bovine mammary epithelial cells (BMECs). However, the regulatory mechanisms of Met in milk protein production in dairy cows remain further clarified. Here, we aimed to investigate the effects of Met on milk protein synthesis in BMECs and explore the underlying mechanism. The effects of Met on the AA transporter, casein synthesis, and the related signaling pathway were evaluated in the BMECs treated with 0.6 mM Met for 6 h combined with or without the inhibition of AA transporter (ASCT2, a neutral AA transporter) activity by the corresponding inhibitor (GPNA). Besides, the effects of SARS on the cells were mainly evaluated in the BMECs treated with 0.6 mM Met for 6 h together with or without SARS knockdown by RNAi interference. The gene expression of AA transporters and pathway-related genes were analyzed by the real-time quantitative polymerase chain reaction method, and the protein expression of related proteins were determined by the western blot assay. Results showed that 0.6 mM Met remarkably enhanced cell growth and ß-casein synthesis compared to the supply of other Met concentrations. Among 13 amino acid transporters, 0.6 mM Met highly increased ASCT2 expression. This Met-stimulated ASCT2 expression and the enhanced mammary intracellular Met uptake were both decreased by the addition of 500 µM GPNA, an inhibitor of ASCT2. In the presence of 0.6 mM Met, the inhibition of ASCT2 activity (by GPNA) and SARS expression (by RNAi) both reduced ß-casein synthesis. Additionally, 0.6 mM Met increased the gene expression of mTOR, S6K1, 4EBP1, and Akt; in contrast, the inhibition of ASCT2 by GPNA lowered the gene expression of these four genes. Collectively, this work suggests that ASCT2 is involved in the SARS-mediated Met stimulation of ß-casein synthesis through enhancing mammary Met uptake and activating the mTOR signaling pathway in BMECs.

Seryl-tRNA synthetase is involved in methionine stimulation of ß-casein synthesis in bovine mammary epithelial cells.

Despite the well-characterised mechanisms of amino acids (AA) regulation of milk protein synthesis in mammary glands (MG), the underlying specific AA regulatory machinery in bovine MG remains further elucidated. As methionine (Met) is one of the most important essential and limiting AA for dairy cows, it is crucial to expand how Met exerts its regulatory effects on dairy milk protein synthesis. Our previous work detected the potential regulatory role of seryl-tRNA synthetase (SARS) in essential AA (EAA)-stimulated bovine casein synthesis. Here, we investigated whether and how SARS participates in Met stimulation of casein production in bovine mammary epithelial cells (BMEC). With or without RNA interference against SARS, BMEC were treated with the medium in the absence (containing all other EAA and devoid of Met alone)/presence (containing 0·6 mm of Met in the medium devoid of Met alone) of Met. The protein abundance of ß-casein and members of the mammalian target of rapamycin (mTOR) and general control nonderepressible 2 (GCN2) pathways was determined by immunoblot assay after 6 h treatment, the cell viability and cell cycle progression were determined by cell counting and propidium iodide-staining assay after 24 h treatment, and protein turnover was determined by l-[ring-3H5]phenylalanine isotope tracing assay after 48 h treatment. In the absence of Met, there was a general reduction in cell viability, total protein synthesis and ß-casein production; in contrast, total protein degradation was enhanced. SARS knockdown strengthened these changes. Finally, SARS may work to promote Met-stimulated ß-casein synthesis via affecting mTOR and GCN2 routes in BMEC.

Structural and Aggregation Features of a Human κ-Casein Fragment with Antitumor and Cell-Penetrating Properties.

Intrinsically disordered proteins play a central role in dynamic regulatory and assembly processes in the cell. Recently, a human κ-casein proteolytic fragment called lactaptin (8.6 kDa) was found to induce apoptosis of human breast adenocarcinoma MCF-7 and MDA-MB-231 cells with no cytotoxic activity toward normal cells. Earlier, we had designed some recombinant analogs of lactaptin and compared their biological activity. Among these analogs, RL2 has the highest antitumor activity, but the amino acid residues and secondary structures that are responsible for RL2's activity remain unclear. To elucidate the structure-activity relations of RL2, we studied the structural and aggregation features of this fairly large intrinsically disordered fragment of human milk κ-casein by a combination of physicochemical methods: NMR, paramagnetic relaxation enhancement (PRE), Electron Paramagnetic Resonance (EPR), circular dichroism, dynamic light scattering, atomic force microscopy, and a cytotoxic activity assay. It was found that in solution, RL2 exists as stand-alone monomeric particles and large aggregates. Whereas the disulfide-bonded homodimer turned out to be more prone to assembly into large aggregates, the monomer predominantly forms single particles. NMR relaxation analysis of spin-labeled RL2 showed that the RL2 N-terminal region, which is essential not only for multimerization of the peptide but also for its proapoptotic action on cancer cells, is more ordered than its C-terminal counterpart and contains a site with a propensity for α-helical secondary structure.

Short communication: Relationship between lysine/methionine ratios and glucose levels and their effects on casein synthesis via activation of the mechanistic target of rapamycin signaling pathway in bovine mammary epithelial cells.

The synthesis of protein requires the availability of specific AA and a large supply of energy in bovine mammary epithelial cells (BMEC). Whether an interaction exists between Lys/Met ratio and glucose level on milk protein synthesis and its potential regulatory mechanism is unclear. We investigated the effects of different Lys/Met ratios and glucose levels on casein synthesis-related gene expression in BMEC to elucidate the underlying molecular mechanisms. Primary BMEC were subjected to 4 treatments for 36 h, arranged in a 2 × 2 factorial design with Lys/Met ratios of 3:1 (1.2:0.4 mM, LM3.0; total AA = 8.24 mM) and 2.3:1 (1.4:0.6 mM, LM2.3; total AA = 8.64 mM) and glucose levels of 17.5 mM (high glucose level) and 2.5 mM (low glucose level). No interactions between Lys/Met ratio and glucose level on cell viability, cell cycle progression, mRNA, or protein expression levels were found. High glucose level increased cell proliferation and promoted cell cycle transition from intermediate phase (G1 phase) to synthesis (S phase) by approximately 50%, whereas Lys/Met ratio had no effect. Both mRNA and protein abundance of αS1-casein and ß-casein were positively affected by LM3.0, whereas a high glucose level increased protein abundance of αS1-casein and ß-casein and increased gene expression of CSN1S1 but not of CSN2. Furthermore, high glucose increased the mRNA abundance of ELF5 and decreased that of GLUT8, enhanced protein expression of total and phosphorylated mechanistic target of rapamycin (mTOR), and decreased phosphorylated AMP-activated protein kinase (AMPK) levels. Treatment LM3.0 had a stimulatory effect on total and phosphorylated mTOR but did not affect AMPK phosphorylation. The mRNA levels of JAK2, ELF5, and RPS6KB1 were upregulated and mRNA levels of EIF4EBP1 were downregulated with LM3.0 compared with LM2.3. Our results indicate that casein synthesis was regulated by Lys/Met ratio via JAK2/ELF5, mTOR, and its downstream RPS6KB1 and EIF4EBP1 signaling. In contrast, glucose regulated casein synthesis through promoting cell proliferation, accelerating cell cycle progression, and activating the ELF5 and AMPK/mTOR signaling pathways. Within the range of substrate levels in the present study, a change in Lys/Met ratio had a stronger effect on abundance of αS1-casein and ß-casein than a change in glucose level.

Sestrin2 Negatively Regulates Casein Synthesis through the SH3BP4-mTORC1 Pathway in Response to AA Depletion or Supplementation in Cow Mammary Epithelial Cells.

Sestrin2 (SESN2) negatively regulates the mammalian target of rapamycin complex 1 (mTORC1) pathway and casein synthesis in response to amino acid (AA) depletion in cow mammary epithelial cells (CMECs); however, the underlying mechanism is unclear. In the current study, the regulation of SESN2 on AA-mediated ß-casein (CSN2) synthesis in CMECs and its mechanism were investigated. Overexpression and silencing of SESN2 demonstrated that SESN2 negatively regulated AA-mediated expression of CSN2 and mTORC1 pathway. Co-immunoprecipitation analysis showed that SESN2 interacted with SH3 domain-binding protein 4 (SH3BP4). Overexpression and silencing of SH3BP4 demonstrated that SH3BP4 negatively regulated AA-mediated expression of CSN2 and mTORC1 pathway and that SESN2 negatively regulated expression of CSN2 and mTORC1 pathway through the SH3BP4 in the presence and absence of AA. The absence or presence of AA demonstrated that AA negatively regulated expression and nuclear localization of activating transcription factor 4 (ATF4). Overexpression and silencing of ATF4 demonstrated that AA negatively regulated SESN2 expression through ATF4. Together, these results indicate that SESN2 negatively regulates the mTORC1 pathway and subsequent CSN2 synthesis through the SH3BP4 in response to AA absence or presence in CMECs.

Proteomic analyses reveal GNG12 regulates cell growth and casein synthesis by activating the Leu-mediated mTORC1 signaling pathway.

In cow mammary epithelial cells (CMECs), cell growth and casein synthesis are regulated by amino acids (AAs), and lysosomes are important organelles in this regulatory process, but the mechanisms remain unclear. Herein, lysosomal membrane proteins (LMPs) in CMECs in the presence (Leu+) and absence (Leu-) of leucine were quantitatively analysed using Sequential Windowed Acquisition of All Theoretical Fragment Ion (SWATH) mass spectrometry. In identified LMPs, Guanine nucleotide-binding protein subunit gamma-12 (GNG12) was a markedly up-regulated protein in Leu+ group. CMECs were treated with Leu+ or Leu-, expression and lysosomal localization of GNG12 were decreased in response to Leu absence. Overexpressing or inhibiting GNG12 demonstrated that cell growth, casein synthesis and activation of the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway were all up-regulated by GNG12. Cell growth, casein synthesis and mTORC1 signaling pathway were decreased in response to Leu absence, but these decreases were partially restored by GNG12 overexpression, and those effects were partially reversed by inhibiting GNG12. Co-immunoprecipitation analysis showed that GNG12 activates the mTORC1 pathway via interaction with Ragulator. Taken together, these results suggest that GNG12 is a positive regulator of the Leu-mediated mTORC1 signaling pathway in CMECs that promotes cell growth and casein synthesis.

Protein 14-3-3ε Regulates Cell Proliferation and Casein Synthesis via PI3K-mTOR Pathway in Dairy Cow Mammary Epithelial Cells.

Cell proliferation and casein synthesis of dairy cow mammary epithelial cells (DCMECs) are regulated by many factors. This research aimed to investigate the effect of 14-3-3ε on cell proliferation and casein synthesis in DCMECs and to reveal the underlying mechanism. Overexpressing or inhibiting 14-3-3ε demonstrated that cell proliferation; casein synthesis; expression of mTOR, p-mTOR, S6K1, and p-S6K1; and lysosomal localization of mTOR were all up-regulated by 14-3-3ε overexpressing and down-regulated by 14-3-3ε inhibiting. In addition, inhibiting mTOR demonstrated that the up-regulation of cell proliferation and casein synthesis in response to 14-3-3ε overexpressing was removed by inhibiting mTOR. Furthermore, the regulatory mechanism of 14-3-3ε was analyzed by coimmunoprecipitation, and we found that 14-3-3ε could interact with PI3K and activate mTORC1 pathway via PI3K. In addition, DCMECs were treated with insulin and prolactin, and the result showed that the cell proliferation and the expression of CSN2 and 14-3-3ε were all up-regulated by these hormones. In conclusion, the current research showed that 14-3-3ε is an important positive regulatory factor for cell proliferation and casein synthesis in DCMECs, as it up-regulates cell proliferation and casein synthesis via activating PI3K-mTOR pathway.

Lysine Stimulates Protein Synthesis by Promoting the Expression of ATB0,+ and Activating the mTOR Pathway in Bovine Mammary Epithelial Cells.

Background: l-lysine (Lys) is a critical dietary nutrient for mammary gland development and milk production. However, the specific pathways of Lys utilization and how milk protein synthesis is affected in bovine mammary epithelial cells (BMECs) are poorly understood. Objective: We aimed to investigate the effects of Lys on milk protein synthesis and the mechanism of Lys uptake and catabolism in BMECs. Methods: BMECs were cultured in 0, 0.5, 1.0, 1.5, 2.0, 5.0, and 10.0 mmol Lys/L to detect cell viability, or cultured in 0-2.0 mmol Lys/L with l-[ring-3H5] phenylalanine to study the effect of Lys on protein turnover, or cultured in Krebs buffer with [U-14C] l-Lys to quantify Lys metabolism. In some experiments, BMECs were cultured in a conditioned medium alone or including 1.0 mmol Lys/L and 2-amino-endo-bicyclo [2.2.1] heptane-2-carboxylic acid (BCH) for 24 h to analyze the expression of amino acid transporter B (0+) (ATB0,+), mammalian target of rapamycin (mTOR), and Janus kinase 2 (JAK2)-signal transducer and activator of transcription 5 (STAT5) pathways. Results: Including 1.0 mmol Lys/L in cultures increased cell viability by 17-47% and protein synthesis by 7-23%, whereas protein degradation was inhibited by 4-64% compared with BMECs cultured with 0, 0.5, or 2.0 mmol Lys/L (all P ≤ 0.05). Studies that used [U-14C] l-Lys showed that most Lys was incorporated into proteins (90%), whereas the remainder was either oxidized into CO2 (4%) or used as a substrate for aspartate (3%) and histidine synthesis (3%). Furthermore, Lys significantly increased expression of ATB0,+ (71% mRNA and 44% protein), STAT5 (27% mRNA and 21% phosphorylated proteins), and mTOR (51% mRNA and 22% phosphorylated proteins) compared with cells without Lys. Conclusions: Lys promoted protein synthesis, mostly through enhancing uptake by ATB0,+ and the mTOR and JAK2-STAT5 pathways. Understanding the utilization of Lys in BMECs provides insights into the role of amino acid nutrition in bovine milk production.

RagD regulates amino acid mediated-casein synthesis and cell proliferation via mTOR signalling in cow mammary epithelial cells.

This research paper addresses the hypothesis that RagD is a key signalling factor that regulates amino acid (AA) mediated-casein synthesis and cell proliferation in cow mammary epithelial cells (CMECs). The expression of RagD was analysed at different times during pregnancy and lactation in bovine mammary tissue from dairy cows. We showed that expression of RagD at lactation period was higher (P < 0·05) than that at pregnancy period. When CMECs were treated with methionine (Met) or lysine (Lys), expression of RagD, ß-casein (CSN2), mTOR and p-mTOR, and cell proliferation were increased. Further, when CMECs were treated to overexpress RagD, expression of CSN2, mTOR and p-mTOR, and cell proliferation were up-regulated. Furthermore, the increase in expression of CSN2, mTOR and p-mTOR, and cell proliferation in response to Met or Lys supply was inhibited by inhibiting RagD, and those effects were reversed in the overexpression model. When CMECs were treated with RagD overexpression together with mTOR inhibition or conversely with RagD inhibition together with mTOR overexpression, results showed that the increase in expression of CSN2 and cell proliferation in response to RagD overexpression was prevented by inhibiting mTOR, and those effects were reversed by overexpressing mTOR. The interaction of RagD with subunit proteins of mTORC1 was analysed, and the result showed that RagD interacted with Raptor. CMECs were treated with Raptor inhibition, and the result showed that the increase in expression of mTOR and p-mTOR in response to RagD overexpression was inhibited by inhibiting Raptor.In conclusion, our study showed that RagD is an important activation factor of mTORC1 in CMECs, activating AA-mediated casein synthesis and cell proliferation, potentially acting via Raptor.

d-Glucose and amino acid deficiency inhibits casein synthesis through JAK2/STAT5 and AMPK/mTOR signaling pathways in mammary epithelial cells of dairy cows.

Amino acids and energy deficiency lead to lower milk protein content in dairy cows. However, the known mechanisms involved in this process do not adequately explain the variability of milk protein concentration in the mammary gland. We hypothesized that a deficiency in d-glucose (d-Glc) or AA would inhibit casein synthesis by regulating signaling pathways in mammary epithelial cells. Cow mammary epithelial cells (CMEC) were subjected to combinations of 1 of 3 concentrations of d-Glc (0, 2.50, or 17.5 mM) and 1 of 3 concentrations of AA (0, 1.03, or 7.20 mM). The effect of each mixture on cell cycle stage was assessed by flow cytometry. The expression levels of ß-casein and κ-casein (encoded by CSN2 and CSN3) were measured by quantitative real-time PCR and Western blotting. Phosphorylation of Janus kinase 2 (Jak2), signal transducer and activator of transcription 5a (Stat5a), AMP-activated protein kinase (AMPK), mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase 1 (S6K1), and eukaryotic factor 4E-binding protein 1 (4EBP1) were analyzed by Western blotting. The percentages of cells in the DNA postsynthetic (G2) and DNA synthesis (S) phases would decrease, with the level of d-Glc or AA declining individually, but no interaction was observed between the d-Glc and AA effects. The CSN2 and CSN3 mRNA and protein were downregulated when d-Glc or AA decreased individually from 17.5 to 2.50 mM or from 7.20 to 1.03 mM, but d-Glc deficiency had a greater effect according to the regression analysis. The phosphorylation ratio of Jak2 (Tyr1007/1008), Stat5a (Tyr694), mTOR (Ser2448), S6K1 (Thr389), and 4EBP1 (Thr37) was downregulated with the level of d-Glc or AA decline, whereas the phosphorylation ratio of AMPK (Thr183/172) was upregulated. And the change of d-Glc level had a more marked effect than AA in regulating the activity of these signaling protein above according to the regression analysis. Thus, d-Glc or AA deficiency likely reduced casein transcription via inhibition of the Jak2/Stat5 pathway, and reduced translation via suppression of the mTOR pathway by activation of AMPK, but d-Glc deficiency had a more marked effect. These indicated that deficiency of AA, and especially Glc, suppressed proliferation of CMEC and casein gene and protein expression, associated with inhibition of JAK2/STAT5 and AMPK/mTOR signaling pathways.

MiR-139 suppresses ß-casein synthesis and proliferation in bovine mammary epithelial cells by targeting the GHR and IGF1R signaling pathways.

BACKGROUND: MicroRNAs have important roles in many biological processes. However, the role of miR-139 in healthy mammary gland remains unclear. The objective of this study was to investigate the effects of miR-139 on lactation in dairy cows. RESULTS: Here, we found that miR-139 was down-regulated in mid-lactation dairy cow mammary tissues compared with mid-pregnancy tissues. Then, we prioritized two of potential target genes of miR-139 in cow, growth hormone receptor (GHR) and type I insulin-like growth factor receptor (IGF1R) for further functional studies based on their roles in lactation processes. Dual luciferase reporter assays validated direct binding of miR-139 to the 3'- untranslated region (UTR) of GHR and IGF1R. Moreover, over-expression or silencing of miR-139 affected mRNA levels of GHR and IGF1R in cultured bovine mammary epithelial cells (BMECs). Furthermore, over-expression of miR-139 decreased protein levels of ß-casein, proliferation in mammary epithelial cell, and the protein levels of IGF1R and key members of the GHR or IGF1R pathways as well, whereas silencing miR-139 produced the opposite result. Among these signal molecules, signal transducer and activator of transcription-5 (STAT5), protein kinase B (also known as AKT1), mammalian target of rapamycin (mTOR), and p70-S6 Kinase (p70S6K) are involed in ß-casein synthesis, and Cyclin D1 is involved in cell proliferation. In addition, silencing GHR decreased protein levels of ß-casein, IGF1R, and key members of the IGF1R pathway, whereas co-silencing miR-139 and GHR rescued the expression of GHR and reversed GHR silencing effects. CONCLUSIONS: Our results demonstrate that GHR and IGF1R are target genes of miR-139 in dairy cow. MiR-139 suppresses ß-casein synthesis and proliferation in BMECs by targeting the GHR and IGF1R signaling pathways.

Optimal ratios of essential amino acids stimulate ß-casein synthesis via activation of the mammalian target of rapamycin signaling pathway in MAC-T cells and bovine mammary tissue explants.

Amino acids are the building blocks of proteins and serve as key molecular components upstream of the signaling pathways that regulate protein synthesis. The objective of this study was to systematically investigate the effect of essential AA ratios on milk protein synthesis in vitro and to elucidate some of the underlying mechanisms. Triplicate cultures of MAC-T cells and bovine mammary tissue explants (MTE) were incubated with the optimal AA ratio (OPAA; Lys:Met, 2.9:1; Thr:Phe, 1.05:1; Lys:Thr, 1.8:1; Lys:His, 2.38:1; and Lys:Val, 1.23:1) in the presence of rapamycin (control), OPAA, a Lys:Thr ratio of 2.1:1, a Lys:Thr ratio of 1.3:1, a Lys:His ratio of 3.05:1, or a Lys:Val ratio of 1.62:1 for 12 h; the other AA concentrations were equal to OPAA. In some experiments, the cells were cultured with OPAA with or without rapamycin (100 ng/mL) or with mammalian target of rapamycin (mTOR) small interference RNA, and the MTE were exposed to OPAA with rapamycin for ß-casein expression. Among the treatments, the expression of ß-casein was greatest in the MTE cultured with OPAA. In MAC-T cells, the OPAA upregulated the mRNA expression of SLC1A5 and SLC7A5 but downregulated the expression of IRS1, AKT3, EEF1A1, and EEF2 compared with the control. The OPAA had no effect on the mTOR phosphorylation status but increased the phosphorylation of S6K1 and RPS6. When the MTE were treated with rapamycin in the presence of OPAA, the expression of ß-casein was markedly decreased. The phosphorylation of RPS6 and 4EBP1 also was reduced in MAC-T cells. A similar negative effect on the expression of RPS6KB1 and EIF4EBP1 was detected when the cells were cultured with either rapamycin or mTOR small interference RNA. The optimal AA ratio stimulated ß-casein expression partly by enhancing the transport of AA into the cells, cross-talk with insulin signaling and a subsequent enhancement of mTOR signaling, or translation elongation in both MAC-T cells and bovine MTE.

Combination of histidine, lysine, methionine, and leucine promotes ß-casein synthesis via the mechanistic target of rapamycin signaling pathway in bovine mammary epithelial cells.

The ratio of different AA in the diets of cows is vital to improve milk protein yield. ß-Casein is one of the important milk proteins with high nutritive value. However, the suitable ratio of essential amino acids (EAA) for the expression of ß-casein in the immortalized bovine mammary epithelial cell line is not fully characterized. This study employed response surface methodology to determine the optimal ratio of His, Lys, Met, and Leu on ß-casein expression level in vitro and clarified the effect of the 4 EAA on ß-casein via the mechanistic target of rapamycin (mTOR) signaling pathway. A central composite design containing 5 axial points per EAA and 28 combinations of the 4 EAA was used in our study. The results of response surface methodology and the changes of the mTOR-related signaling proteins were determined by western blot. The results showed that ß-casein level was significantly affected by all 4 EAA (R2 = 0.71). The optimum conditions for ß-casein expression are found to be 5.47 mM of His, 7.48 mM of Lys, 1.17 mM of Met, and 8.21 mM of Leu (His:Lys:Met:Leu = 5:6:1:7) in the designed scope of concentration. The interaction of Leu and Met significantly affected ß-casein expression (P < 0.01). The phosphorylation of mTOR (Ser2481), regulatory associated protein of target of rapamycin (Ser792), ribosomal protein S6 kinase 1 (Thr389), ribosomal protein S6 (Ser235/236), and eukaryotic elongation factor 2 (Thr56) was increased with the supplementation of either single EAA or an optimal combination of EAA. However, the phosphorylation of eukaryotic initiation factor 4E binding protein 1 (Thr37) was decreased with the addition of Lys, Met, or Leu alone. Furthermore, the phosphorylation (P) of eIF2α (Ser51) was decreased when Met was supplemented alone. Under the optimal mixture of 4 EAA, the phosphorylation of mechanistic target of rapamycin complex 1 signaling proteins was significantly greater than the single EAA supplementations and the expression of ß-casein was 98% as high as the positive control (i.e., medium with all AA). A similar trend was found with P-ribosomal protein S6 kinase 1 and P-ribosomal protein S6. In conclusion, the extracellular concentrations of His, Lys, Met, and Leu at a ratio of 5:6:1:7 maximized ß-casein expression in the immortalized bovine mammary epithelial cell line may occur via activation of the mechanistic target of rapamycin complex 1 signaling pathway.

Production of Recombinant Antimicrobial Polymeric Protein Beta Casein-E 50-52 and Its Antimicrobial Synergistic Effects Assessment with Thymol.

Accelerating emergence of antimicrobial resistance among food pathogens and consumers' increasing demands for preservative-free foods are two contemporary challenging aspects within the food industry. Antimicrobial packaging and the use of natural preservatives are promising solutions. In the present study, we used beta-casein-one of the primary self-assembly proteins in milk with a high polymeric film production capability-as a fusion partner for the recombinant expression of E 50-52 antimicrobial peptide in Escherichia coli. The pET21a-BCN-E 50-52 construct was transformed to E. coli BL21 (DE3), and protein expression was induced under optimized conditions. Purified protein obtained from nickel affinity chromatography was refolded under optimized dialysis circumstances and concentrated to 1600 µg/mL fusion protein by ultrafiltration. Antimicrobial activities of recombinant BCN-E 50-52 performed against Escherichia coli, Salmonella typhimurium, Listeria monocytogenes, Staphylococcus aureus, Aspergillus flavus, and Candida albicans. Subsequently, the synergistic effects of BCN-E 50-52 and thymol were assayed. Results of checkerboard tests showed strong synergistic activity between two compounds. Time-kill and growth kinetic studies indicated a sharp reduction of cell viability during the first period of exposure, and SEM (scanning electron microscope) results validated the severe destructive effects of BCN E 50-52 and thymol in combination on bacterial cells.

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

The main purpose of this work was to determine the effect of arginase inhibition via Nω-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.

Response surface methodology applied to the generation of casein hydrolysates with antioxidant and dipeptidyl peptidase IV inhibitory properties.

BACKGROUND: Hydrolysis parameters affecting the release of dipeptidyl peptidase IV (DPP-IV) inhibitory and antioxidant peptides from milk proteins have not been extensively studied. Therefore, a multifactorial (i.e. pH, temperature and hydrolysis time) composite design was used to optimise the release of bioactive peptides (BAPs) with DPP-IV inhibitory and antioxidant [oxygen radical absorbance capacity (ORAC)] properties from sodium caseinate. RESULTS: Fifteen sodium caseinate hydrolysates (H1-H15) were generated with ProtamexTM , a bacillus proteinase activity. Hydrolysis time (1 to 5 h) had the highest influence on both DPP-IV inhibitory properties and ORAC activity (P < 0.05). Alteration of incubation temperature (40 to 60 °C) and pH (6.5 to 8.0) had an effect on the DPP-IV inhibitory properties but not the ORAC activity of the Protamex sodium caseinate hydrolysates. A multi-functional hydrolysate, H12, was identified having DPP-IV inhibitory (actual: 0.82 ± 0.24 vs. predicted optimum: 0.68 mg mL-1 ) and ORAC (actual: 639 ± 66 vs. predicted optimum: 639 µmol TE g-1 ) activity of the same order (P > 0.05) as the response surface methodology (RSM) predicted optimum bioactivities. CONCLUSION: Generation of milk protein hydrolysates through multifactorial design approaches may aid in the optimal enzymatic release of BAPs with serum glucose lowering and antioxidant properties. © 2016 Society of Chemical Industry.

Engineering of double recombinant vaccinia virus with enhanced oncolytic potential for solid tumor virotherapy.

Vaccinia virus (VACV) oncolytic therapy has been successful in a number of tumor models. In this study our goal was to generate a double recombinant vaccinia virus (VV-GMCSF-Lact) with enhanced antitumor activity that expresses exogenous proteins: the antitumor protein lactaptin and human granulocyte-macrophage colony-stimulating factor (GM-CSF). Lactaptin has previously been demonstrated to act as a tumor suppressor in mouse hepatoma as well as MDA-MB-231 human adenocarcinoma cells grafted into SCID mice. VV-GMCSF-Lact was engineered from Lister strain (L-IVP) vaccinia virus and has deletions of the viral thymidine kinase and vaccinia growth factor genes. Cell culture experiments revealed that engineered VV-GMCSF-Lact induced the death of cultured cancer cells more efficiently than recombinant VACV coding only GM-CSF (VV-GMCSF-dGF). Normal human MCF-10A cells were resistant to both recombinants up to 10 PFU/cell. The selectivity index for breast cancer cells measured in pair cultures MCF-7/MCF-10A was 200 for recombinant VV-GMCSF-Lact coding lactaptin and 100 for VV-GMCSF-dGF. Using flow cytometry we demonstrated that both recombinants induced apoptosis in treated cells but that the rate in the cells with active caspase-3 and -7 was higher after treatment with VV-GMCSF-Lact than with VV-GMCSF-dGF. Tumor growth inhibition and survival outcomes after VV-GMCSF-Lact treatment were estimated using immunodeficient and immunocompetent mice models. We observed that VV-GMCSF-Lact efficiently delays the growth of sensitive and chemoresistant tumors. These results demonstrate that recombinant VACVs coding an apoptosis-inducing protein have good therapeutic potential against chemoresistant tumors. Our data will also stimulate further investigation of coding lactaptin double recombinant VACV in clinical settings.