scholarly journals PCV2 Triggers PK-15 Cell Apoptosis Through the PLC–IP3R–Ca2+ Signaling Pathway

2021 ◽  
Vol 12 ◽  
Author(s):  
Shuo Wang ◽  
Chen Li ◽  
Panpan Sun ◽  
Jianli Shi ◽  
Xiaoyan Wu ◽  
...  
Keyword(s):  
Virus Infection ◽  
Signaling Pathway ◽  
Cell Apoptosis ◽  
Protein Biosynthesis ◽  
Quantitative Polymerase Chain Reaction ◽  
Pcv2 Infection ◽  
Porcine Circovirus ◽  
Intracellular Ca ◽  
Polymerase Chain ◽  
Trisphosphate Receptor

The endoplasmic reticulum (ER) plays an essential role in Ca2+ concentration balance and protein biosynthesis. During infection, the virus needs to complete its life process with the help of ER. At the same time, ER also produces ER stress (ERS), which induces apoptosis to resist virus infection. Our study explored the Ca2+ concentration, ERS, and the apoptosis mechanism after porcine circovirus 2 (PCV2) infection. We show here that PCV2 infection induces the increased cytoplasmic Ca2+ level and PK-15 cell ER swelling. The colocalization of phospholipase C (PLC) and inositol 1,4,5-trisphosphate receptor (IP3R) in the cytoplasm was observed by laser confocal microscopy. Western blot and quantitative polymerase chain reaction experiments confirmed that PLC and IP3R expression levels increased after PCV2 infection, and Ca2+ concentration in the cytoplasm increased after virus infection. These results suggest that PCV2 infection triggers ERS of PK-15 cells via the PLC–IP3R–Ca2+ signaling pathway to promote the release of intracellular Ca2+ and led to cell apoptosis.

scholarly journals PCV2 trigger apoptosis of PK-15 cells through the PLC-IP3R-Ca2+ signaling pathway

2020 ◽  
Author(s):  
Shuo Wang ◽  
Chen Li ◽  
Panpan Sun ◽  
Jianli Shi ◽  
Xiaoyan Wu ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Viral Infection ◽  
Signaling Pathway ◽  
Cell Apoptosis ◽  
Porcine Circovirus Type ◽  
Pcv2 Infection ◽  
Porcine Circovirus ◽  
Cellular Processes ◽  
Key Enzyme ◽  
Phosphatidylinositol 4

Phospholipase C (PLC) is a key enzyme in the cell membrane. PLC hydrolyses phosphatidylinositol 4, 5-bisphosphate (PIP2) to generateinositol 1,4, 5-triphosphate (IP3) and diacylglycerol (DAG) that regulates a variety of cellular processes. Evidence indicates the pivotal role of PLC and inositol 1,4,5-trisphosphate receptor(IP3R) in influencing Ca2+ release from the endoplasmic reticulum(ER).At the same time, the imbalance of Ca2+ will stimulate endoplasmic reticulum stress(ERS), leading to cell apoptosis. Viral infection could triggers host defense through apoptosis of the infected cells.However, it is not clear how porcine circovirus type 2 (PCV2) induces apoptosis by affecting Ca2+ homeostasis. We show here that PCV2 infection induces the increased cytoplasmic Ca2+ level and apoptosis.We also found that the ER swelling of PK-15 cells after viral infection by transmission electron microscopy. Furthemore, the activation of PLC-IP3R-Ca2+ signaling enhanced apoptosis in infected PK-15 cells. Taken together,our findings suggest that PCV2 infection trigger ERS of PK-15 cells via the PLC-IP3R-Ca2+ signaling pathway to promoted the release of intracellular Ca2+, and led to cell apoptosis.

scholarly journals Transcript Profiles of Blumeria graminis Development During Infection Reveal a Cluster of Genes That Are Potential Virulence Determinants

2005 ◽  
Vol 18 (2) ◽  
pp. 125-133 ◽  
Author(s):  
Maike Both ◽  
Sabine E. Eckert ◽  
Michael Csukai ◽  
Elisabeth Müller ◽  
George Dimopoulos ◽  
...  
Keyword(s):  
Protein Biosynthesis ◽  
Pathogenic Fungus ◽  
Quantitative Polymerase Chain Reaction ◽  
Blumeria Graminis ◽  
Host Cells ◽  
Virulence Determinants ◽  
Polymerase Chain ◽  
Transcript Profiles ◽  
Encoding Protein ◽  
Late Stages

High-density cDNA microarrays (2,027 unigenes) were used to analyze transcript profiles of the plant-pathogenic fungus Blumeria graminis f. sp. hordei throughout its asexual life cycle and development of infection. RNA was obtained from four stages preceding penetration and four stages after penetration of the host cells. The microarray data was validated by comparing the expression of a plasma membrane H+-ATPase and fructose-1,6-bis phosphatase with the data obtained from a quantitative polymerase chain reaction (PCR) assay. The results showed that there was a global switch in expression between the pre- and postpenetrative stages. This was largely due to accumulation of RNA encoding protein biosynthesis genes in the late stages. Other functional clusters, such as virulence-related genes and sterol metabolism genes, are up-regulated in pre- and postpenetration stages, respectively. A group of RNAs whose abundance correlated with the expression of cap20, a gene known to be required for virulence in Colletotrichum gloeosporioides, identified genes that are strong candidates for pathogenicity factors in B. graminis.

scholarly journals NLRX1 can counteract innate immune response induced by an external stimulus favoring HBV infection by competitive inhibition of MAVS-RLRs signaling in HepG2-NTCP cells

2021 ◽  
Vol 104 (4) ◽  
pp. 003685042110580
Author(s):  
Qian Jiao ◽  
Wenxiong Xu ◽  
Xiaoyan Guo ◽  
Huiyuan Liu ◽  
Baolin Liao ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Virus Infection ◽  
Hepatitis B ◽  
Competitive Inhibition ◽  
Quantitative Polymerase Chain Reaction ◽  
Innate Immune ◽  
Hepatitis B Virus Infection ◽  
B Virus ◽  
Polymerase Chain ◽  
External Stimuli

Introduction This study is aimed at the determination of the effect of the immune-regulatory factor NLRX1 on the antiviral activity of hepatocytes against an external stimuli favoring hepatitis B virus infection, and to explore its mechanism of action. Methods A HepG2-NTCP model was established using the LV003 lentivirus. Cells were transfected using an overexpression vector and NLRX1 siRNA to achieve overexpression and interference of NLRX1 expression (OV-NLRX1, si-NLRX1). Levels of HBsAg and HBcAg were determined using Western blotting analysis and immunohistochemical analysis. The levels of hepatitis B virus DNA and hepatitis B virus cccDNA were determined by real-time quantitative polymerase chain reaction. The expression and transcriptional activity of IFN-α, IFN-β, and IL-6 were measured using real-time quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, and promoter-luciferase reporter plasmids. Co-immunoprecipitation was used to determine the effect of NLRX1 on the interaction between MAVS and RIG-1. Western blotting was used to obtain the phosphorylation of essential proteins in the MAVS-RLRs signaling pathways. Results NLRX1 promoted HepG2-NTCP cell hepatitis B virus infection. Compared to the control group, the levels of HBsAg, HBcAg, hepatitis B virus cccDNA, and hepatitis B virus DNA increased in the OV-NLRX1 group and decreased in the si-NLRX1. Co-immunoprecipitation results showed that NLRX1 competitively inhibited the interaction between MAVS and RIG-1, and inhibited the phosphorylation of p65, IRF3, and IRF7. Additionally, NLRX1 reduced the transcription activity and expression levels of the final products: IFN-α, IFN-β, and IL-6. Conclusions NLRX1 can counteract innate immune response induced by an external stimuli favoring hepatitis B virus infection by competitive inhibition of MAVS-RLRs signaling in HepG2-NTCP cells. Inhibition of the MAVS-RLR-mediated signaling pathways leads to a decline in the expression levels of I-IFN and IL-6.

scholarly journals Torque teno sus virus 1 and 2 viral loads in faeces of porcine circovirus 2-positive pigs

2015 ◽  
Vol 84 (2) ◽  
pp. 91-95 ◽  
Author(s):  
Alessandra M. M. G. de Castro ◽  
Cintia M. Baldin ◽  
Cintia M. Favero ◽  
Priscilla F. Gerber ◽  
Rafael I. Cipullo ◽  
...  
Keyword(s):  
Quantitative Polymerase Chain Reaction ◽  
Porcine Circovirus ◽  
Age Group ◽  
Nursery Pigs ◽  
Viral Loads ◽  
Faecal Samples ◽  
Disease Condition ◽  
Porcine Circovirus 2 ◽  
Polymerase Chain ◽  
Torque Teno Sus Virus

Recently, studies have suggested an association between the Torque teno sus virus (TTSuV) and the Porcine circovirus-2 (PCV2) in PCV2-associated disease cases. The aim of this study was to verify TTSuVs loads in pig faeces from PCV2-positive animals with and without diarrhea from PCVAD-affected and PCV2-unvaccinated herds. A total of 80 faecal samples were collected individually from nursery and grow-finish pigs with (n = 40) or without (n = 40) diarrhea. The samples were tested for PCV2 and TTSuVs by using DNA binding dye SYBR Green quantitative polymerase chain reaction (qPCR). Torque teno sus virus k2 (TTSuVk2) load in the faeces was significantly higher in the nursery pigs with diarrhea, and these pigs also exhibited significantly higher PCV2 (P < 0.01) faecal matter loads compared to the non-diarrheic animals from the same age group. Torque teno sus virus 1 (TTSuV1) viral loads were the same regardless of age group and disease condition. There were no correlations between PCV2 and TTSuV1 or TTSuVk2 and TTSuV1 viral loads; however, a weak correlation (r = 0.23, P = 0.03) was found between TTSuVk2 and PCV2 viral loads. In conclusion, TTSuVk2 viral loads were significantly higher in the diarrheic faeces from the nursery pigs. Additionally, the higher loads of PCV2 and TTSuVk2 in the nursery-diarrheic animals revealed that diarrhea might have an important role in the spread of both viruses in herds.

Establishment and Application of Dual TaqMan Fluorescence Quantitative Polymerase Chain Reaction (PCR) for the Detection of Porcine Circovirus Types 2 and 3

2021 ◽  
Author(s):  
Fengyang Cao ◽  
Xiaoning Luan ◽  
Wenwen Wang ◽  
Yuyan Li ◽  
Longzong Guo ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Genetic Variation ◽  
Quantitative Polymerase Chain Reaction ◽  
Shandong Province ◽  
Whole Genome Sequence ◽  
Porcine Circovirus ◽  
Whole Genome ◽  
Chain Reaction ◽  
Positive Rate ◽  
Polymerase Chain

Abstract To establish a rapid method for the simultaneous differential detection of porcine circovirus types 2 (PCV2) and 3 (PCV3), two pairs of primers and two TaqMan probes were designed according to the gene sequences of PCV2 and PCV3, and a dual TaqMan fluorescence quantitative polymerase chain reaction (PCR) method for the simultaneous detection of two virus nucleic acids was established. The results showed that the correlation coefficients (R2) of the standard curves drawn using the recombinant plasmids of PCV2 and PCV3 were greater than 0.99 and had a good linear relationship. The specific detection results of PCV type 1, Porcine parvovirus, and Porcine pseudorabies virus were negative. The detection limits of this experimental method for PCV2 and PCV3 were 10 and 1 copies/μL, respectively, which were more sensitive than those of the common PCR detection methods. The established method was used to detect 76 samples from some pig farms in Shandong Province. 11 of the 76 samples were PCV3 positive (positive rate of 14.47%), 24 were PCV2 positive (positive rate of 34.58%), and PCV3 and PCV2 were mixed in six samples (positive rate of 7.89%). The whole-genome sequence of PCV3 was amplified and sequenced to further understand the molecular biological characteristics and genetic variation of PCV3 in Shandong Province. The genomes of 11 PCV3 strains were all 2000 bp long, and the whole-genome sequence homology between them ranged from 98.4% to 99.9%. There were mutation sites in the amino acid sequences of Cap and Rep proteins, and the strains isolated in this experiment were concentrated in the PCV3a and PCV3c subgroups. This study provides technical support and molecular biological basis for nucleic acid detection, epidemiological characteristics, genetic variation, and control of PCV3 in Shandong Province.

scholarly journals Sulforaphane Protect Against Cadmium-Induced Oxidative Damage in mouse Leydigs cells by Activating Nrf2/ARE Signaling Pathway

2019 ◽  
Vol 20 (3) ◽  
pp. 630 ◽  
Author(s):  
Shu-Hua Yang ◽  
Peng Li ◽  
Li-Hui Yu ◽  
Lin Li ◽  
Miao Long ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Mrna Expression ◽  
Signaling Pathway ◽  
Cell Apoptosis ◽  
Antioxidant Response ◽  
Enzyme Linked Immunosorbent Assay ◽  
Related Factor ◽  
Expression Of Genes ◽  
Glutamylcysteine Synthetase ◽  
Polymerase Chain

Cadmium (Cd) is harmful for humans and animals, especially for the reproductive system. However, the mechanism of its toxicity has not been elucidated, and how to alleviate its toxicity is very important. This study aimed to explore the role and mechanism of action of sulforaphane (SFN) in protecting mouse Leydigs (TM3) cells from cadmium (Cd)-induced damage. The half-maximal inhibitory concentration (IC50) of Cd and the safe doses of SFN were determined using a methyl thiazolyl tetrazolium (MTT) assay. The testosterone secretion from TM3 cells was measured using the enzyme-linked immunosorbent assay. The intracellular oxidative stress was evaluated using corresponding kits. The cell apoptosis was detected using flow cytometry. The mRNA expression of genes associated with NF-E2-related factor 2 (Nrf2)/antioxidant response element (ARE) signaling was detected using reverse transcription–polymerase chain reaction, including Nrf2, heme oxygenase I (HO-1), glutathione peroxidase (GSH-Px), NAD(P)H:quinone acceptor oxidoreductase 1 (NQO1), and γ-glutamylcysteine synthetase (γ-GCS). The protein expression of Nrf2, GSH-Px, HO-1, γ-GCS, and NQO1 was detected using Western blot analysis. The results showed that the IC50 of Cd to TM3 cells was 51.4 µmol/L. SFN reduced the release of lactate dehydrogenase from Cd-exposed cells. Cd + SFN 2.5 treatment significantly elevated testosterone concentration compared with the Cd group (p < 0.05). SFN significantly increased total superoxide dismutase (T-SOD) and GSH-Px activity and GSH content in Cd-treated cells (p < 0.05; p < 0.01), inhibited the production of malondialdehyde or reactive oxygen species caused by Cd (p < 0.05; p < 0.01), and reduced the apoptotic rate of Cd-induced TM3 cells (p < 0.01). SFN upregulated the mRNA expression of Nrf2, GSH-Px, HO-1, NQO1, and γ-GCS in Cd-treated cells, indicating the protective effect of SFN against Cd-induced oxidative stress or cell apoptosis by activating the Nrf2/ARE signaling pathway.

Detection of porcine circovirus in rodents — Short communication

2010 ◽  
Vol 58 (2) ◽  
pp. 265-268 ◽  
Author(s):  
Márta Lőrincz ◽  
Attila Cságola ◽  
Imre Biksi ◽  
Levente Szeredi ◽  
Ádám Dán ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Short Communication ◽  
Pcv2 Infection ◽  
Porcine Circovirus ◽  
Chain Reaction ◽  
Wild Boars ◽  
Pig Farms ◽  
Porcine Circoviruses ◽  
Polymerase Chain

Porcine circoviruses (PCV) are present worldwide, infecting domestic pigs and wild boars alike. Studies under laboratory conditions indicated that PCV can be taken up by mice and the virus can replicate in these animals. The possible role of rodents in maintaining and transmitting PCV2 infection in the field has not been investigated yet. The present study reports the detection of PCV2, the pathogenic form of the virus, in mice and rats. A number of rodents, such as mice, rats and voles, were collected at PCV2-infected farms and also outside pig herds and tested for the presence of the virus by polymerase chain reaction (PCR). The results indicated that PCV2 can be present both in mice and rats (65.0% and 23.8% positivity, respectively) on the infected premises, but those rodents that were collected outside pig farms remained negative for PCV2.

scholarly journals Astragalus polysaccharides protects thapsigargin-induced endoplasmic reticulum stress in HT29 cells

2019 ◽  
Vol 14 (1) ◽  
pp. 494-501
Author(s):  
Lie Zheng ◽  
Ya-Li Zhang ◽  
Xuan Chen ◽  
De-Liang Chen ◽  
Yan-Cheng Dai ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Signaling Pathway ◽  
Quantitative Polymerase Chain Reaction ◽  
Mrna Levels ◽  
Chain Reaction ◽  
Gene Expressions ◽  
Astragalus Polysaccharides ◽  
Ht29 Cells ◽  
Polymerase Chain

AbstractAimThis study investigates the effect of astragalus polysaccharides (APS) in protecting against thapsigargin-induced endoplasmic reticulum (ER) stress in HT29 cells by suppressing the PERK-eIF2a signaling pathway.MethodsHT29 cells were induced by thapsigargin for 12 hours, then treated with APS for 24 hours, and the gene expressions of GRP78, CHOP and eIF2a were quantified by reverse transcription quantitative polymerase chain reaction (RT-qPCR). The expression of GRP78, CHOP, PERK, p-PERK, eIF2a, and p-eIF2a were detected by Western blot.ResultsThe ER stress caused by thapsigargin strongly up-regulated the expression of GRP78 and CHOP in HT29 cells, which activated the PERK-eIF2a pathway. There was an increase in PERK phosphorylation, and induction of eIF2a in HT29 cells. Thapsigargin caused significant ER expansion in HT29 cells due to the 12-hour ER stress. Importantly, Astragalus polysaccharide significantly inhibited the phosphorylation of PERK and eIF2a, which reduced the mRNA levels of GRP78, CHOP, PERK and eIF2a, and inhibited the ER expansion in HT29 cells after 24 hours of treatment.ConclusionThe results indicate that APS reduces the expression of GRP78 and CHOP in HT29 cells, at least in part, by preventing the activation of the PERK-eIF2a signaling pathway.

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