scholarly journals Dipeptide (Methionyl-Methionine) Transport and Its Effect on β-Casein Synthesis in Bovine Mammary Epithelial Cells

2018 ◽  
Vol 49 (2) ◽  
pp. 479-488 ◽  
Author(s):  
Caihong Wang ◽  
Fengqi Zhao ◽  
Jianxin Liu ◽  
Hongyun Liu
Keyword(s):  
Epithelial Cells ◽  
Cell Viability ◽  
Transport Properties ◽  
Mammary Epithelial Cells ◽  
Mammary Epithelial ◽  
Janus Kinase ◽  
Initiation Factor ◽  
Peptide Transporter ◽  
Small Interference Rna ◽  
Bovine Mammary Epithelial Cells

Background/Aims: The aim of this study was to investigate the transport properties and utilization of methionyl-methionine dipeptide (Met-Met) in β-casein (β-CN) synthesis in bovine mammary epithelial cells (BMECs). Methods: The transport properties were studied for the effects of time, pH, concentration, temperature and inhibitors using Met-Met-FITC in BMECs. BMECs were treated with different concentrations of Met-Met (0, 20, 40, 80, 120 and 160 µg/ml). In several experiments, the cells were treated with Janus kinase 2 (JAK2) inhibitor (tyrphostin AG-490, 50 µM) and mammalian target of rapamycin (mTOR) inhibitor (rapamycin, 100 ng/ml). Results: The uptake of Met-Met-FITC by BMECs was rapid during the first fifteen minutes and became saturated after 15 minutes. The transport of Met-Met-FITC in BMECs exhibited a Michaelis constant of 52.4 µM and maximum transport velocity of 14.8 pmol/min/mg protein. The uptake of Met-Met-FITC in BMECs was pH-dependent, peaked at pH 6.5 and was significantly inhibited by other peptides, including Met-Lys, Lys-Lys, Gly-Met, Gly-Leu and Met-Leu. Knocking down the peptide transporter 2 (PepT2) with small interference RNA markedly decreased Met-Met-FITC uptake. Met-Met concentration-dependently increased the PepT2 expression and β-CN synthesis in BMECs with an optimal concentration of 80 µg/ml. At 80 µg/ml, Met-Met also enhanced the cell viability and cyclin D1 expression and promoted cell cycle transition from G1 phase to S phase. In addition, 80 µg/ml Met-Met increased the mRNA abundance of JAK2 and signal transducer and activator of transcription 5 (STAT5) and enhanced the phosphorylation of JAK2, STAT5, mTOR, p70 ribosomal S6 kinase 1 and eukaryotic initiation factor 4E binding protein 1. The inhibition of JAK2 and mTOR significantly decreased Met-Met-induced increase in cell viability and β-CN synthesis in BMECs. Conclusion: Our data elucidated the properties of peptide transporter and its effect on β-CN synthesis in BMECs. Met-Met, taken up by PepT2, enhances cell proliferation and promotes β-CN synthesis by activating JAK2-STAT5 and mTOR signaling pathways in BMECs.

scholarly journals Caffeic Acid Prevented LPS-Induced Injury of Primary Bovine Mammary Epithelial Cells through Inhibiting NF-κB and MAPK Activation

2019 ◽  
Vol 2019 ◽  
pp. 1-12 ◽  
Author(s):  
Mingjiang Liu ◽  
Guoqing Fang ◽  
Shaojie Yin ◽  
Xin Zhao ◽  
Chi Zhang ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cell Viability ◽  
Signaling Pathways ◽  
Caffeic Acid ◽  
Proinflammatory Cytokines ◽  
Mammary Epithelial Cells ◽  
Mammary Epithelial ◽  
Signal Pathways ◽  
Dependent Manner ◽  
Bovine Mammary Epithelial Cells

In our previous study, lipopolysaccharide (LPS) significantly reduced the cell viability of primary bovine mammary epithelial cells (bMEC) leading to cell apoptosis, which were prevented by caffeic acid (CA) through inhibiting NF-κB activation and reducing proinflammatory cytokine expression. While the underlying mechanism remains unclear, here, we determined that LPS induced the extensive microstructural damage of bMEC, especially the mitochondria and endoplasmic reticulum. Then, the obvious reduction of mitochondrial membrane potential and expression changes of apoptosis-associated proteins (Bcl-2, Bax, and casepase-3) indicated that apoptosis signaling through the mitochondria should be responsible for the cell viability decrease. Next, the high-throughput cDNA sequencing (RNA-Seq) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were employed to verify that the MAPK and JAK-STAT signaling pathways also were the principal targets of LPS. Following, the critical proteins (ERK, JNK, p38, and c-jun) of the MAPK signaling pathways were activated, and the release of proinflammatory cytokines (TNF-α, IL-1β, IL-6, and IL-8) regulated by NF-κB and MAPKs was significantly increased, which can promote a cascade of inflammation that induces cell injury and apoptosis. Meanwhile, CA significantly inhibited the activation of MAPKs and the release of proinflammatory cytokines in a dose-dependent manner, which were similar to its effects on the NF-κB activation that we previously published. So we concluded that CA regulates the proteins located in the upstream of multiple cell signal pathways which can reduce the LPS-induced activation of NF-κB and MAPKs, thus weakening the inflammatory response and maintaining cell structure and function, which accordingly inhibit apoptosis.

14-3-3γaffects eIF5 to regulateβ-casein synthesis in bovine mammary epithelial cells

2016 ◽  
Vol 96 (4) ◽  
pp. 478-487
Author(s):  
Cuiping Yu ◽  
Chaochao Luo ◽  
Xinyu Gu ◽  
Yanli Zang ◽  
Bo Qu ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Regulatory Mechanism ◽  
Mammary Epithelial Cells ◽  
Mammary Epithelial ◽  
Initiation Factor ◽  
Biological Processes ◽  
Bovine Mammary Epithelial Cells ◽  
Protein Levels ◽  
Casein Synthesis

The 14-3-3γ protein participates in many biological processes; however, its regulatory mechanism in milk protein synthesis is not well studied. We hypothesized that 14-3-3γ might affect eIF5 (an initiation factor) to regulate β-casein synthesis in dairy cows. In this study, a possible interaction between 14-3-3γ and eIF5 was investigated using bovine mammary epithelial cells (BMECs). The expression levels of 14-3-3γ and eIF5 in the mammary gland tissues from cows producing higher quality milk were higher than those from cows producing low-quality milk. Moreover, the expression of 14-3-3γ, eIF5, and β-casein were increased at both mRNA and protein levels in BMECs cultured in vitro with methionine (Met) supplementation. Coimmunoprecipitation, colocalization, and FRET analysis further showed the evidences that 14-3-3γ physically bound to eIF5 in BMECs. Gene function studies revealed that 14-3-3γ positively regulated eIF5 through alteration of eIF2α/p-eIF2α ratio. Collectively, our data suggest that 14-3-3γ regulates β-casein translation in BMECs through interaction with eIF5.

scholarly journals Effects of Chinese Propolis in Protecting Bovine Mammary Epithelial Cells against Mastitis Pathogens-Induced Cell Damage

2016 ◽  
Vol 2016 ◽  
pp. 1-12 ◽  
Author(s):  
Kai Wang ◽  
Xiao-Lu Jin ◽  
Xiao-Ge Shen ◽  
Li-Ping Sun ◽  
Li-Ming Wu ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cell Viability ◽  
Inflammatory Responses ◽  
Mammary Epithelial Cells ◽  
Cell Damage ◽  
Mammary Epithelial ◽  
Active Components ◽  
Antioxidant Genes ◽  
Bovine Mammary Epithelial Cells ◽  
Chinese Propolis

Chinese propolis (CP), an important hive product, can alleviate inflammatory responses. However, little is known regarding the potential of propolis treatment for mastitis control. To investigate the anti-inflammatory effects of CP on bovine mammary epithelial cells (MAC-T), we used a range of pathogens to induce cellular inflammatory damage. Cell viability was determined and expressions of inflammatory/antioxidant genes were measured. Using a cell-based reporter assay system, we evaluated CP and its primary constituents on the NF-κB and Nrf2-ARE transcription activation. MAC-T cells treated with bacterial endotoxin (lipopolysaccharide, LPS), heat-inactivatedEscherichia coli,andStaphylococcus aureusexhibited significant decreases in cell viability while TNF-αand lipoteichoic acid (LTA) did not. Pretreatment with CP prevented losses in cell viability associated with the addition of killed bacteria or bacterial endotoxins. There were also corresponding decreases in expressions of proinflammatory IL-6 and TNF-αmRNA. Compared with the mastitis challenged cells, enhanced expressions of antioxidant genes HO-1, Txnrd-1, and GCLM were observed in CP-treated cells. CP and its polyphenolic active components (primarily caffeic acid phenethyl ester and quercetin) had strong inhibitive effects against NF-κB activation and increased the transcriptional activity of Nrf2-ARE. These findings suggest that propolis may be valuable in the control of bovine mastitis.

scholarly journals 5-ALA Attenuates the Palmitic Acid-Induced ER Stress and Apoptosis in Bovine Mammary Epithelial Cells

Molecules ◽  
2021 ◽  
Vol 26 (4) ◽  
pp. 1183
Author(s):  
Mst Mamuna Sharmin ◽  
Md Aminul Islam ◽  
Itsuki Yamamoto ◽  
Shin Taniguchi ◽  
Shinichi Yonekura
Keyword(s):  
T Cells ◽  
Epithelial Cells ◽  
Palmitic Acid ◽  
Er Stress ◽  
Aminolevulinic Acid ◽  
Mammary Epithelial Cells ◽  
Mammary Epithelial ◽  
Protective Effects ◽  
Bovine Mammary Epithelial Cells ◽  
Induced Apoptosis

The conservation of mammary gland physiology by maintaining the maximum number of mammary epithelial cells (MECs) is of the utmost importance for the optimum amount of milk production. In a state of negative energy balance, palmitic acid (PA) reduces the number of bovine MECs. However, there is no effective strategy against PA-induced apoptosis of MECs. In the present study, 5-aminolevulinic acid (5-ALA) was established as a remedial agent against PA-induced apoptosis of MAC-T cells (an established line of bovine MECs). In PA-treated cells, the apoptosis-related genes BCL2 and BAX were down- and upregulated, respectively. The elevated expression of major genes of the unfolded protein response (UPR), such as CHOP, a proapoptotic marker (C/EBP homologous protein), reduced the viability of PA-treated MAC-T cells. In contrast, 5-ALA pretreatment increased and decreased BCL2 and BAX expression, respectively. Moreover, cleaved caspase-3 protein expression was significantly reduced in the 5-ALA-pretreated group in comparison with the PA group. The downregulation of major UPR-related genes, including CHOP, extended the viability of MAC-T cells pretreated with 5-ALA and also reduced the enhanced intensity of the PA-induced expression of phospho-protein kinase R-like ER kinase. Moreover, the enhanced expression of HO-1 (antioxidant gene heme oxygenase) by 5-ALA reduced PA-induced oxidative stress (OxS). HO-1 is not only protective against OxS but also effective against ER stress. Collectively, these findings offer new insights into the protective effects of 5-ALA against PA-induced apoptosis of bovine MECs.

Exogenous phospholipase A2 affects inflammatory gene expression in primary bovine mammary epithelial cells

2019 ◽  
Vol 86 (2) ◽  
pp. 177-180
Author(s):  
Jacqueline P. Kurz ◽  
Mark P. Richards ◽  
Matthew Garcia ◽  
Zhongde Wang
Keyword(s):  
Epithelial Cells ◽  
Phospholipase A2 ◽  
Inflammatory Responses ◽  
Mammary Epithelial Cells ◽  
Constitutive Expression ◽  
Mammary Epithelial ◽  
Inflammatory Factors ◽  
Bovine Mammary Epithelial Cells ◽  
Lps Challenge

AbstractThis Research Communication addresses the hypothesis that exogenously administered phospholipase A2 (PLA2) affects the inflammatory responses of bovine mammary epithelial cells (bMEC) in vitro with the aim of providing preliminary justification of investigation into the uses of exogenously administered PLA2 to manage or treat bovine mastitis. Primary bMEC lines from 11 lactating Holstein dairy cows were established and the expression of 14 pro-inflammatory genes compared under unchallenged and lipopolysaccharide (LPS)-challenged conditions, with and without concurrent treatment with bovine pancreatic PLA2G1B, a secreted form of PLA2. No differences in the expression of these genes were noted between PLA2-treated and untreated bMEC under unchallenged conditions. Following LPS challenge, untreated bMEC exhibited significant downregulation of CXCL8, IL1B, CCL20, and CXCL1. In contrast, PLA2-treated bMEC exhibited significant downregulation of IL1B and CCL20 only. These findings indicate that exogenous PLA2 affects the expression of some pro-inflammatory factors in immune-stimulated bMEC, but does not influence the constitutive expression of these factors. Further investigation of the influence of exogenous PLA2 in the bovine mammary gland is justified.

Cell cycle regulation of mammary epithelial cell detachment byStaphylococcus aureus

1996 ◽  
Vol 63 (4) ◽  
pp. 543-553 ◽  
Author(s):  
Boris Zavizion ◽  
Andrew J. Bramley ◽  
Ioannis Politis
Keyword(s):  
Cell Cycle ◽  
Epithelial Cells ◽  
Culture Medium ◽  
Mammary Epithelial Cells ◽  
Mammary Epithelial ◽  
Cell Detachment ◽  
Staph Aureus ◽  
Bovine Mammary Epithelial Cells ◽  
Isogenic Mutants ◽  
Cycle Regulation

SummaryThe effect ofStaphylococcus aureuson detachment of bovine mammary epithelial cells in culture was examined. Mammary epithelial cells became detached from fresh monolayers following a 3 h incubation in the presence ofStaph. aureusM60. Two different procedures indicated that cell detachment coincided with the S-phase of the cell cycle. The roles of proteinases, toxins and Ca availability in inducing cell detachment were examined. Addition of the proteinase inhibitor phenyl-methylsulphonyl fluoride (1 mM) to the culture medium prevented cell detachment. Addition of a combination of purified staphylococcal proteinases XVI and XVII-B to the culture medium of mammary epithelial cells induced cell detachment in the absence ofStaph. aureus. Cell detachment may be caused by a staphylococcal proteinase. However, addition of Ca (10 mM) to the culture medium abolishedStaph. aureus-induced cell detachment, despite the fact that proteinase activity was still apparently present. Isogenic mutants ofStaph. aureusM60, expressing either ± or β toxins but not both, induced cell detachment, but to a lesser extent than the wild type. Thus, Ca and toxins play some role during cell detachment. Clones established from detached cells that were washed and replated showed the same susceptibility toStaph. aureus-induced cell detachment as the parental cells. This indicated that there is no subclone of mammary epithelial cells more sensitive to this effect.

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