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Establishment of a Madin-Darby bovine kidney cell line expressing anchorless bovine prion protein.

ABSTRACT: Enzyme-linked immunosorbent assay (ELISA) performed using extensively purified bacterially expressed bovine prion protein (PrP) shows decreased cross-reactivity. We generated a transduced Madin-Darby bovine kidney (MDBK) cell line continuously expressing glycosylphosphatidylinositol (GPI)-anchorless bovine PrP (designated as MDBK ?GPI protein) by using a lentiviral expression system. The present study also described the method for purifying bovine PrP through sequential culturing without the need for complex purification protocol. Our results showed that the purified bovine PrP could be used as an immunogen for developing anti-PrP monoclonal antibodies. Together, our results suggest that the new GPI-anchorless bovine PrP and its purification method can be used for performing basic studies for employing a cell-based approach.

SUBMITTER: Kim HJ

PROVIDER: S-EPMC6021889 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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Establishment of a Madin-Darby bovine kidney cell line expressing anchorless bovine prion protein.

Kim Hyo-Jin HJ Roh In-Soon IS Park Hoo-Chang HC Ahn Su Bi SB Suh Tae-Young TY Park Kyung-Je KJ Kang Hae-Eun HE Sohn Hyun-Joo HJ

The Journal of veterinary medical science 20180404 6

Enzyme-linked immunosorbent assay (ELISA) performed using extensively purified bacterially expressed bovine prion protein (PrP) shows decreased cross-reactivity. We generated a transduced Madin-Darby bovine kidney (MDBK) cell line continuously expressing glycosylphosphatidylinositol (GPI)-anchorless bovine PrP (designated as MDBK ∆GPI protein) by using a lentiviral expression system. The present study also described the method for purifying bovine PrP through sequential culturing without the nee ...[more]

PMID: 29618668

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Zinc supplementation protects against cadmium accumulation and cytotoxicity in Madin-Darby bovine kidney cells.

Project description:Cadmium ions (Cd2+) have been reported to accumulate in bovine tissues, although Cd2+ cytotoxicity has not been investigated thoroughly in this species. Zinc ions (Zn2+) have been shown to antagonize the toxic effects of heavy metals such as Cd2+ in some systems. The present study investigated Cd2+ cytotoxicity in Madin-Darby bovine kidney (MDBK) epithelial cells, and explored whether this was modified by Zn2+. Exposure to Cd2+ led to a dose- and time-dependent increase in apoptotic cell death, with increased intracellular levels of reactive oxygen species and mitochondrial damage. Zn2+ supplementation alleviated Cd2+-induced cytotoxicity and this protective effect was more obvious when cells were exposed to a lower concentration of Cd2+ (10 ?M), as compared to 50 ?M Cd2+. This indicated that high levels of Cd2+ accumulation might induce irreversible damage in bovine kidney cells. Metallothioneins (MTs) are metal-binding proteins that play an essential role in heavy metal ion detoxification. We found that co-exposure to Zn2+ and Cd2+ synergistically enhanced RNA and protein expression of MT-1, MT-2, and the metal-regulatory transcription factor 1 in MDBK cells. Notably, addition of Zn2+ reduced the amounts of cytosolic Cd2+ detected following MDBK exposure to 10 ?M Cd2+. These findings revealed a protective role of Zn2+ in counteracting Cd2+ uptake and toxicity in MDBK cells, indicating that this approach may provide a means to protect livestock from excessive Cd2+ accumulation.

| S-EPMC4126686 | biostudies-literature

Effect of bovine viral diarrhoea virus infection in Madin-Darby bovine kidney cells

Project description:Analysis of gene expression induced in Madin-Darby bovine kidney cells (MDBK) infected with Bovine viral diarrhoea virus BVDV2 strain 1373. Keywords: other

2004-05-31 | GSE756 | GEO

Bovine herpes virus type 1 induces apoptosis through Fas-dependent and mitochondria-controlled manner in Madin-Darby bovine kidney cells.

Project description:BackgroundBovine herpesvirus type 1 (BHV-1) is an important pathogen in cattle that is responsible for substantial economic losses. Previous studies suggest that BHV-1 may induce apoptosis in Madin-Darby bovine kidney (MDBK) cells via a mechanism only involving caspases and p53. However, the mechanism for BHV-1-induced MDBK cell apoptosis still requires more research.MethodsMDBK was used as a model to study the precise signaling pathways of apoptosis induced by BHV-1 infection.ResultsBHV-1 infection activated a Fas/FasL-mediated apoptotic pathway, resulting in activation of caspase-8 and cleavage of Bid. In addition, BHV-1 infection down-regulated Bcl-2 and up-regulated Bax expression, thereby initiating the release of cytochrome c followed by caspase-9 activation. The combined activation of the extrinsic and intrinsic pathways resulted in activation of downstream effecter caspase-3 and poly ADP-ribose polymerase (PARP), leading to apoptosis. Furthermore, blocking apoptosis using caspase inhibitors improved BHV-1-infected MDBK cell viability to different extent. BHV-1 infection did not induce significant DNA fragmentation in MDBK cells pretreated with ammonium chloride (NH4Cl) or cells infected with UV-inactivated BHV-1. Blocking caspases activation increased BHV-1 replication.ConclusionsBHV-1 induces apoptosis in MDBK cells through extrinsic and intrinsic pathways and there might be cross-talk between the two pathways. In addition, BHV-1 replication may be necessary for the induction of apoptosis in BHV-1-infected cells, and prolonged cell viability benefits BHV-1 replication.

| S-EPMC3502331 | biostudies-literature

High-Throughput MicroRNA Profiles of Permissive Madin-Darby Canine Kidney Cell Line Infected with Influenza B Viruses.

Project description:Victoria and Yamagata lineages of influenza B viruses are globally circulating in seasonal epidemics. Madin-Darby canine kidney (MDCK) cells are permissive for viral isolation and vaccine manufacture. Nevertheless, the interplay between influenza B viruses and host microRNAs has not been investigated in this cell line. Therefore, the present study aims at high-throughput analysis of canine microRNA profile upon infection of influenza B viruses. Briefly, MDCK cells were infected with Victoria or Yamagata lineage at MOI of 0.01. After being harvested at 6, 12 and 24 h post infection, microRNAs were subjected to high-throughput sequencing based on MiSeq platform (Illumina). The results demonstrated that five microRNAs including cfa-miR-197, cfa-miR-215, cfa-miR361, cfa-miR-1841, and cfa-miR-1842 were overexpressed in both Victoria and Yamagata lineage infections. Interestingly, computational prediction showed that karyopherin alpha 6 (KPNA6) was targeted by cfa-miR-197 and cfa-miR-215. Moreover, the binding sites of both microRNAs were assessed by 3'-UTR reporter assay. The results showed that only cfa-miR-197 could bind to the target sites of KPNA6, leading to suppressing luciferase activity. Additionally, silencing of KPNA6 was confirmed by overexpression of cfa-miR-197. This study provides canine microRNA responses to seasonal influenza B viruses, suggesting that virus-mediated microRNAs might play crucial roles in host gene regulation.

| S-EPMC6893747 | biostudies-literature

Analysis of microRNAs Expression Profiles in Madin-Darby Bovine Kidney Cells Infected With Caprine Parainfluenza Virus Type 3.

Project description:Caprine parainfluenza virus type 3 (CPIV3) is a newly emerging pathogenic respiratory agent infecting both young and adult goats, and it was identified in eastern China in 2013. Cellular microRNAs (miRNAs) have been reported to be important modulators of the intricate virus-host interactions. In order to elucidate the role of miRNAs in madin-darby bovine kidney (MDBK) cells during CPIV3 infection. In this study, we performed high-throughput sequencing technology to analyze small RNA libraries in CPIV3-infected and mock-infected MDBK cells. The results showed that a total of 249 known and 152 novel candidate miRNAs were differentially expressed in MDBK cells after CPIV3 infection, and 22,981 and 22,572 target genes were predicted, respectively. In addition, RT-qPCR assay was used to further confirm the expression patterns of 13 of these differentially expressed miRNAs and their mRNA targets. Functional annotation analysis showed these up- and downregulated target genes were mainly involved in MAPK signaling pathway, Jak-STAT signaling pathway, Toll-like receptor signaling pathway, p53 signaling pathway, focal adhesion, NF-kappa B signaling pathway, and apoptosis, et al. To our knowledge, this is the first report of the comparative expression of miRNAs in MDBK cells after CPIV3 infection. Our finding provides information concerning miRNAs expression profile in response to CPIV3 infection, and offers clues for identifying potential candidates for antiviral therapies against CPIV3.

| S-EPMC5885596 | biostudies-literature

Effect of bovine viral diarrhoea virus infection in Madin-Darby bovine kidney cells

Project description:Analysis of gene expression induced in Madin-Darby bovine kidney cells (MDBK) infected with Bovine viral diarrhoea virus BVDV2 strain 1373. Keywords: other

2004-05-31 | E-GEOD-756 | biostudies-arrayexpress

Depletion of E-cadherin disrupts establishment but not maintenance of cell junctions in Madin-Darby canine kidney epithelial cells.

Project description:E-cadherin forms calcium-dependent homophilic intercellular adhesions between epithelial cells. These contacts regulate multiple aspects of cell behavior, including the organization of intercellular tight junctions (TJs). To distinguish between the roles of E-cadherin in formation versus maintenance of junctions, Madin-Darby canine kidney (MDCK) cells were depleted of E-cadherin by RNA interference. Surprisingly, reducing E-cadherin expression had little effect on the protein levels or localization of adherens junction (AJ) or TJ markers. The cells underwent morphological changes, as the normally flat apical surface swelled into a dome. However, apical-basal polarity was not compromised, transmembrane resistance was normal, and zonula occludin protein 1 dynamics at the TJs were unchanged. Additionally, an E-cadherin/Cadherin-6 double knockdown also failed to disrupt established TJs, although beta-catenin was lost from the cell cortex. Nevertheless, cells depleted of E-cadherin failed to properly reestablish cell polarity after junction disassembly. Recovery of cell-cell adhesion, transepithelial resistance, and the localization of TJ and AJ markers were all delayed. In contrast, depletion of alpha-catenin caused long-term disruption of junctions. These results indicate that E-cadherin and Cadherin-6 function as a scaffold for the construction of polarized structures, and they become largely dispensable in mature junctions, whereas alpha-catenin is essential for the maintenance of functional junctions.

| S-EPMC1751327 | biostudies-literature

Madin-Darby canine kidney cells expression array

Project description:cDNA expression array of epithelial phenotype untreated MDCK cells; after 30 days of TGFb treatment, which induces a mesenchymal phenotype; TGF-b-treated cells that are treated with 5-AZA for 72 h; and 30 days after TGFb withdrawal Gene expression was analyzed in a genome-wide manner, to asses those changes occurring upon TGF-b-induced epithelial to mesenchymal transition (EMT), those that are stimulated by treatment with a demethylating agent, and which are restored after TGF-b withdrawal

2014-08-14 | GSE48525 | GEO

STAT1 is required for redifferentiation during Madin-Darby canine kidney tubulogenesis.

Project description:Tubule formation in vitro using Madin-Darby canine kidney (MDCK) epithelial cells consists mainly of two processes. First, the cells undergo a partial epithelial-mesenchymal transition (pEMT), losing polarity and migrating. Second, the cells redifferentiate, forming cords and then tubules with continuous lumens. We have shown previously that extracellular signal-regulated kinase activation is required for pEMT. However, the mechanism of how the pEMT phase is turned off and the redifferentiation phase is initiated is largely unknown. To address the central question of the sequential control of these two phases, we used MDCK cells grown as cysts and treated with hepatocyte growth factor to model tubulogenesis. We show that signal transducer and activator of transcription (STAT)1 controls the sequential progression from the pEMT phase to the redifferentiation phase. Loss of STAT1 prevents redifferentiation. Constitutively active STAT1 allows redifferentiation to occur even when cells are otherwise prevented from progressing beyond the pEMT phase by exogenous activation of Raf. Moreover, tyrosine phosphorylation defective STAT1 partially restored cord formation in such cells, suggesting that STAT1 functions in part as nonnuclear protein mediating signal transduction in this process. Constitutively active or inactive forms of STAT1 did not promote lumen maturation, suggesting this requires a distinct signal.

| S-EPMC2982126 | biostudies-literature

Sodium butyrate induced gene expression changes in the Madin-Darby bovine kidney epithelial cells

Project description:The Madin-Darby bovine kidney epithelial cells, when at approximately 50% confluence (during the exponential phase), were treated with 10mM sodium butyrate for 24h. Butyrate induced profound changes in gene expression related with multiple signal transduction pathways such as cell cycle control, apoptosis and extracellular matrix remodeling in this cell line Keywords: stress response, two class unpaired design

2006-01-05 | GSE3970 | GEO

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