Molecular characterization and protective efficacy of a new conserved hypothetical protein of Eimeria tenella

Eimeria tenella is an obligate intracellular parasite that actively invades cecal epithelial cells of chickens. This parasite encodes a genome of more than 8000 genes. However, more than 70% of the gene models for this species are currently annotated as hypothetical proteins. In this study, a conserved hypothetical protein gene of E. tenella, designated EtCHP18905, was cloned and identified, and its immune protective effects were evaluated. The open reading frame of EtCHP18905 was 1053bp and encoded a protein of 350 amino acids with a molecular weight of 38.7kDa. The recombinant EtCHP18905 protein (rEtCHP18905) was expressed in E. coli. Using western blot, the recombinant protein was successfully recognized by anti GST-Tag monoclonal antibody and anti-sporozoites protein rabbit serum. Real-time quantitative PCR analysis revealed that the EtCHP18905 mRNA levels were higher in sporozoites than in unsporulated oocysts, sporulated oocysts and second-generation merozoites. Western blot analysis showed that EtCHP18905 protein expression levels were lower in sporozoites than in other stages. Immunofluorescence analysis indicated that the EtCHP18905 protein was located on the surface of sporozoites and second-generation merozoites. Inhibition experiments showed that the ability of sporozoites to invade host cells was significantly decreased after treatment with the anti-rEtCHP18905 polyclonal antibody. Vaccination with rEtCHP18905 protein was able to significantly decrease mean lesion scores and oocyst outputs as compared to non-vaccinated controls. The results suggest that the rEtCHP18905 protein can induce partial immune protection against infection with E. tenella and could be an effective candidate for the development of new vaccines.

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Apoptotic effects of antimalarial artemisinin on the second generation merozoites of Eimeria tenella and parasitized host cells

Veterinary Parasitology ◽

10.1016/j.vetpar.2014.09.025 ◽

2014 ◽

Vol 206 (3-4) ◽

pp. 297-303 ◽

Cited By ~ 4

Author(s):

Pinghua Mo ◽

Qingtao Ma ◽

Xin Zhao ◽

Nan Cheng ◽

Jianping Tao ◽

...

Keyword(s):

Second Generation ◽

Eimeria Tenella ◽

Host Cells ◽

Apoptotic Effects

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Epigallocatechingallate Inhibits Migration of Human Uveal Melanoma Cells via Downregulation of Matrix Metalloproteinase-2 Activity and ERK1/2 Pathway

BioMed Research International ◽

10.1155/2014/141582 ◽

2014 ◽

Vol 2014 ◽

pp. 1-9 ◽

Cited By ~ 18

Author(s):

Chi-Wu Chang ◽

Yi-Hsien Hsieh ◽

Wei-En Yang ◽

Shun-Fa Yang ◽

Yueqin Chen ◽

...

Keyword(s):

Western Blot ◽

Uveal Melanoma ◽

Gelatin Zymography ◽

Melanoma Cells ◽

Matrix Metalloproteinase 2 ◽

Pcr Analysis ◽

Signal Pathways ◽

Mrna Levels ◽

Dependent Manner ◽

Uveal Melanoma Cell

The effects of epigallocatechingallate (EGCG) on the migration and expression of MMP-2 of uveal melanoma cells have not been reported. We studied this effect and relevant signaling pathways in a human uveal melanoma cell line (M17). MTT study found that EGCG did not affect the cell viability of M17 cells up to 100 µM. Wound-healing assay showed that EGCG significantly reduced the migration of melanoma cells in a dose-dependent manner from 20 to 100 µM. Gelatin zymography showed that secreted MMP-2 activity was dose-dependently inhibited by EGCG, whereas the MMP-2 expression at protein and mRNA levels was not affected as determined by western blot and RT-PCR analysis. EGCG significantly increased the expressions of MMP-2 endogenous inhibitors (TIMP-2 and RECK) in M17 cells. Western blot analysis of MAPK signal pathways showed that EGCG significantly decreased phosphorylated ERK1/2 levels, but not p38 and JNK levels, in melanoma cells. ERK1/2 inhibitors also reduced the migration and activity of MMP-2 in M17 cells. The present study suggested EGCG at nontoxic levels could inhibit migration of melanoma cells via downregulation of activities of secreted MMP-2 through the inhibition of the ERK1/2 phosphorylation. Therefore, EGCG may be a promising agent to be explored for the prevention of metastasis of uveal melanoma.

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Secretoneurin is a potential paracrine factor from lactotrophs stimulating gonadotropin release in the goldfish pituitary

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00407.2010 ◽

2010 ◽

Vol 299 (5) ◽

pp. R1290-R1297 ◽

Cited By ~ 11

Author(s):

E. Zhao ◽

Caleb L. Grey ◽

Dapeng Zhang ◽

Jan A. Mennigen ◽

Ajoy Basak ◽

...

Keyword(s):

Gene Expression ◽

Western Blot ◽

Blot Analysis ◽

Western Blot Analysis ◽

Time Course ◽

Pcr Analysis ◽

Pituitary Cells ◽

Mrna Levels ◽

Paracrine Factor ◽

Lh Release

Secretoneurin (SN) is a functional neuropeptide derived from the evolutionarily conserved part of precursor protein secretogranin II (SgII). In the time course study, SN (10 nM) stimulates luteinizing hormone (LH) production and secretion after 6 h of static incubation of goldfish pituitary cells. Due to the existence of SN-immunoreactivity (SN-IR) in goldfish lactotrophs, endogenous SN might exert a paracrine effect on LH in the pituitary. In an in vitro immunoneutralization experiment, coincubation with anti-SN antiserum reduces the stimulatory effect of salmon gonadotropin-releasing hormone (sGnRH) on LH release by 64%. Using Western blot analysis, we demonstrate that sGnRH significantly increases the expression of the major SgII-derived peptide (∼57 kDa, with SN-IR) and prolactin (PRL) after 12 h in the static culture of goldfish pituitary cells. Furthermore, there exists a significant correlation between the levels of these two proteins ( R = 0.76, P = 0.004). Another ∼30 kDa SgII-derived peptide containing SN is only observed in sGnRH-treated pituitary cells. Consistent with the Western blot analysis results, real-time RT-PCR analysis shows that a 12-h treatment with sGnRH induced 1.6- and 1.7-fold increments in SgII and PRL mRNA levels, respectively. SgII gene expression was also associated with PRL gene expression ( R = 0.66; P = 0.02). PRL cells loaded with the calcium-sensitive dye, fura 2/AM, respond to sGnRH treatment with increases in intracellular Ca2+ concentration level, suggesting a potential mechanism of GnRH on PRL cells and thus SgII processing and SN secretion. Taken together, endogenous lactotroph-generated SN, under the control of hypothalamic GnRH, exerts a paracrine action on neighboring gonadotrophs to stimulate LH release.

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Molecular characterization and protective efficacy of the microneme 2 protein from Eimeria tenella

Parasite ◽

10.1051/parasite/2018061 ◽

2018 ◽

Vol 25 ◽

pp. 60 ◽

Cited By ~ 10

Author(s):

Ming Yan ◽

Xiaoxia Cui ◽

Qiping Zhao ◽

Shunhai Zhu ◽

Bing Huang ◽

...

Keyword(s):

Protective Efficacy ◽

Body Weight Gain ◽

Expression System ◽

Protozoan Parasite ◽

Eimeria Tenella ◽

Host Cells ◽

Apicomplexan Parasites ◽

The Mean ◽

First And Second Generation ◽

Average Body

Microneme proteins play an important role in the adherence of apicomplexan parasites to host cells during the invasion process. In this study, the microneme 2 protein from the protozoan parasite Eimeria tenella (EtMIC2) was cloned, characterized, and its protective efficacy as a DNA vaccine investigated. The EtMIC2 gene, which codes for a 35.07 kDa protein in E. tenella sporulated oocysts, was cloned and recombinant EtMIC2 protein (rEtMIC2) was produced in an Escherichia coli expression system. Immunostaining with an anti-rEtMIC2 antibody showed that the EtMIC2 protein mainly localized in the anterior region and membrane of sporozoites, in the cytoplasm of first- and second-generation merozoites, and was strongly expressed during first-stage schizogony. In addition, incubation with specific antibodies against EtMIC2 was found to efficiently reduce the ability of E. tenella sporozoites to invade host cells. Furthermore, animal-challenge experiments demonstrated that immunization with pcDNA3.1(+)-EtMIC2 significantly increased average body weight gain, while decreasing the mean lesion score and oocyst output in chickens. Taken together, these results suggest that EtMIC2 plays an important role in parasite cell invasion and may be a viable candidate for the development of new vaccines against E. tenella infection in chickens.

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The P2Y1 Receptor in the Colonic Submucosa of Rats and Its Correlation with Opioid-Induced Constipation

10.21203/rs.3.rs-892072/v1 ◽

2021 ◽

Author(s):

Yuqiong Zhao ◽

Xiaojie Ren ◽

Fan Li ◽

Binghan Jia ◽

Dengke Wang ◽

...

Keyword(s):

Protein Expression ◽

Western Blot ◽

Pcr Analysis ◽

Mrna Levels ◽

Submucosal Plexus ◽

Rt Pcr ◽

Selective Agonist ◽

Fecal Water ◽

Μ Opioid Receptor ◽

Relative Mrna Expression

Abstract Aims: To explore the expression changes of P2Y1 in the distal colonic submucosa of opioid induced constipation (OIC) rats and its correlation with the occurrence of OIC. Methods: OIC model was generated by intraperitoneal injection of loperamide hydrochloride, a selective agonist of the μ-opioid receptor (MOR). Seven days later, the model was assessing by detecting the fecal traits and calculating the fecal water cotent. The distribution of MOR-containing neurons and P2Y1-containing neurons in colonic submucosal plexus of rat were demonstrated by immunofluorescence histochemistry. Western Blot was used to evaluate the expression changes of MOR, P2Y1 and ATP synthase subunit beta (ATPB) in colonic submucosa, while the RT-PCR analysis was performed to determine the relative mRNA expression of MOR, P2Y1 and ATPB. Results: After seven days, the feces of OIC rats had an appearance of like sausage-shaped pieces, and the fecal water content, stool weight of OIC rats were decreased. Immunofluorescence histochemistry showed the co-expression of MOR and ATPB, P2Y1 and calbindin (CB) in the nerve cells of distal colonic submucosal plexus. RT-PCR showed that MOR mRNA levels were significantly increased in the distal colonic submucosa of OIC rats, while the mRNA levels of P2Y1 were decreased. Western blot results showed that MOR protein expression was increased, and the P2Y1 protein expression was significantly decreased in the distal colonic submucosa of OIC rats.Conclusion: P2Y1 is associated with the occurrence of OIC in rats, and the expression of MOR and P2Y1 and OIC are correlated with each other.

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Phenotypical and Functional Characteristics of Human Regulatory T Cells during Ex Vivo Maturation from CD4+ T Lymphocytes

Applied Sciences ◽

10.3390/app11135776 ◽

2021 ◽

Vol 11 (13) ◽

pp. 5776

Author(s):

Varvara G. Blinova ◽

Natalia S. Novachly ◽

Sofya N. Gippius ◽

Abdullah Hilal ◽

Yulia A. Gladilina ◽

...

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Ex Vivo ◽

Pcr Analysis ◽

Mrna Levels ◽

Rt Pcr ◽

Inflammatory Reactions ◽

Effector Cells ◽

Cycle Regulation ◽

Further Development

Regulatory T cells (Tregs) participate in the negative regulation of inflammatory reactions by suppressing effector cells. In a number of autoimmune disorders, the suppressive function and/or the number of Tregs is compromised. The lack of active functioning Tregs can be restored with adoptive transfer of expanded ex vivo autologous Tregs. In our study, we traced the differentiation and maturation of Tregs CD4+CD25+FoxP3+CD127low over 7 days of cultivation from initial CD4+ T cells under ex vivo conditions. The resulting ex vivo expanded cell population (eTregs) demonstrated the immune profile of Tregs with an increased capacity to suppress the proliferation of target effector cells. The expression of the FoxP3 gene was upregulated within the time of expansion and was associated with gradual demethylation in the promotor region of the T cell-specific demethylation region. Real-time RT-PCR analysis revealed changes in the expression profile of genes involved in cell cycle regulation. In addition to FOXP3, the cells displayed elevated mRNA levels of Ikaros zinc finger transcription factors and the main telomerase catalytic subunit hTERT. Alternative splicing of FoxP3, hTERT and IKZF family members was demonstrated to be involved in eTreg maturation. Our data indicate that expanded ex vivo eTregs develop a Treg-specific phenotype and functional suppressive activity. We suggest that eTregs are not just expanded but transformed cells with enhanced capacities of immune suppression. Our findings may influence further development of cell immunosuppressive therapy based on regulatory T cells.

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Activation of Apoptosis in a βB1-CTGF Transgenic Mouse Model

International Journal of Molecular Sciences ◽

10.3390/ijms22041997 ◽

2021 ◽

Vol 22 (4) ◽

pp. 1997

Author(s):

Maximilian Weiss ◽

Sabrina Reinehr ◽

Ana M. Mueller-Buehl ◽

Johanna D. Doerner ◽

Rudolf Fuchshofer ◽

...

Keyword(s):

Open Angle Glaucoma ◽

Bipolar Cells ◽

Nerve Degeneration ◽

Terminal Deoxynucleotidyl Transferase ◽

Pcr Analysis ◽

Progression Rate ◽

Suitable Model ◽

Mrna Levels ◽

Gabaergic Synapse ◽

Slow Progression

To reveal the pathomechanisms of glaucoma, a common cause of blindness, suitable animal models are needed. As previously shown, retinal ganglion cell and optic nerve degeneration occur in βB1-CTGF mice. Here, we aimed to determine possible apoptotic mechanisms and degeneration of different retinal cells. Hence, retinae were processed for immunohistology (n = 5–9/group) and quantitative real-time PCR analysis (n = 5–7/group) in 5- and 10-week-old βB1-CTGF and wildtype controls. We noted significantly more cleaved caspase 3+ cells in βB1-CTGF retinae at 5 (p = 0.005) and 10 weeks (p = 0.02), and a significant upregulation of Casp3 and Bax/Bcl2 mRNA levels (p < 0.05). Furthermore, more terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL+) cells were detected in transgenic mice at 5 (p = 0.03) and 10 weeks (p = 0.02). Neurofilament H staining (p = 0.01) as well as Nefh (p = 0.02) and Tubb3 (p = 0.009) mRNA levels were significantly decreased at 10 weeks. GABAergic synapse intensity was lower at 5 weeks, while no alterations were noted at 10 weeks. The glutamatergic synapse intensity was decreased at 5 (p = 0.007) and 10 weeks (p = 0.01). No changes were observed for bipolar cells, photoreceptors, and macroglia. We conclude that apoptotic processes and synapse loss precede neuronal death in this model. This slow progression rate makes the βB1-CTGF mice a suitable model to study primary open-angle glaucoma.

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GMP-compliant fully automated radiosynthesis of [18F]FEPPA for PET/MRI imaging of regional brain TSPO expression

EJNMMI Research ◽

10.1186/s13550-021-00768-9 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Chi-Wei Chang ◽

Chuang-Hsin Chiu ◽

Ming-Hsien Lin ◽

Hung-Ming Wu ◽

Tsung-Hsun Yu ◽

...

Keyword(s):

Mr Imaging ◽

Western Blotting ◽

Second Generation ◽

Mrna Levels ◽

Automated Radiosynthesis ◽

Lps Challenge ◽

Tnf Α ◽

The Brain ◽

Tspo Expression

Abstract Background Expression of translocator protein (TSPO) on the outer mitochondrial membrane of activated microglia is strongly associated with neuroinflammation. The second-generation PET ligand [18F]FEPPA specifically binds TSPO to enable in vivo visualization and quantification of neuroinflammation. We optimized a fully automated radiosynthesis method and evaluated the utility of [18F]FEPPA, the second-generation PET ligand specifically binds TSPO, in a mouse model of systemic LPS challenge to detect TSPO-associated signals of central and peripheral inflammation. In vivo dynamic PET/MR imaging was performed in LPS-induced and control mice after [18F]FEPPA administration. The relationship between the [18F]FEPPA signal and the dose of LPS was assessed. The cytokine levels (i.e., TNF-α, Il-1β, Il-6) in LPS-induced mice were measured by RT-PCR. Standard uptake value (SUV), total volume of distribution (VT) and area under the curve (AUC) were determined based on the metabolite-uncorrected plasma input function. Western blotting and immunostaining were used to measure TSPO expression in the brain. Results The fully automated [18F]FEPPA radiosynthesis produced an uncorrected radiochemical yield of 30 ± 2% within 80 min, with a radiochemical purity greater than 99% and specific activity of 148.9‒216.8 GBq/µmol. Significant differences were observed in the brain after [18F]FEPPA administration: SUV, VT and AUC were 1.61 ± 0.1, 1.25 ± 0.12 and 1.58 ± 0.09-fold higher in LPS-injected mice than controls. TNF-α, Il-1β and Il-6 mRNA levels were also elevated in the brains of LPS-injected mice. Western blotting revealed TSPO (p < 0.05) and Iba-1 (p < 0.01) were upregulated in the brain after LPS administration. In LPS-injected mice, TSPO immunoactivity colocalized with Iba-1 in the cerebrum and TSPO was significantly overexpressed in the hippocampus and cerebellum. The peripheral organs (heart, lung) of LPS-injected mice had higher [18F]FEPPA signal-to-noise ratios than control mice. Conclusions Based on the current data on ligand specificity and selectivity in central tissues using 7 T PET/MR imaging, we demonstrate that [18F]FEPPA accumulations significant increased in the specific brain regions of systemic LPS-induced neuroinflammation (5 mg/kg). Future investigations are needed to determine the sensitivity of [18F]FEPPA as a biomarker of neuroinflammation as well as the correlation between the PET signal intensity and the expression levels of TSPO.

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Cell Culture-Derived Tilapia Lake Virus-Inactivated Vaccine Containing Montanide Adjuvant Provides High Protection against Viral Challenge for Tilapia

Vaccines ◽

10.3390/vaccines9020086 ◽

2021 ◽

Vol 9 (2) ◽

pp. 86

Author(s):

Weiwei Zeng ◽

Yingying Wang ◽

Huzi Hu ◽

Qing Wang ◽

Sven M. Bergmann ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Protective Efficacy ◽

Interferon Γ ◽

Economic Losses ◽

Mrna Levels ◽

Infectious Dose ◽

Booster Immunization ◽

Virus Challenge ◽

High Level ◽

Factor Α

Tilapia lake virus (TiLV) is a newly emerging pathogen responsible for high mortality and economic losses in the global tilapia industry. Currently, no antiviral therapy or vaccines are available for the control of this disease. The goal of the present study was to evaluate the immunological effects and protective efficacy of formaldehyde- and β-propiolactone-inactivated vaccines against TiLV in the presence and absence of the Montanide IMS 1312 VG adjuvant in tilapia. We found that β-propiolactone inactivation of viral particles generated a vaccine with a higher protection efficacy against virus challenge than did formaldehyde. The relative percent survivals of vaccinated fish at doses of 108, 107, and 106 50% tissue culture infectious dose (TCID50)/mL were 42.9%, 28.5%, and 14.3% in the absence of the adjuvant and 85.7%, 64.3%, and 32.1% in its presence, respectively. The vaccine generated specific IgM and neutralizing antibodies against TiLV at 3 weeks following immunization that were significantly increased after a second booster immunization. The steady state mRNA levels of the genes tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interferon γ (IFN-γ), cluster of differentiation 4 (CD4), major histocompatibility complex (MHC)-Ia, and MHC-II were all increased and indicated successful immune stimulation against TiLV. The vaccine also significantly lowered the viral loads and resulted in significant increases in survival, indicating that the vaccine may also inhibit viral proliferation as well as stimulate a protective antibody response. The β-propiolactone-inactivated TiLV vaccine coupled with the adjuvant Montanide IMS 1312 VG and booster immunizations can provide a high level of protection from virus challenge in tilapia.

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Pulchinenosides from Pulsatilla Chinensis Increase P-Glycoprotein Activity and Induce P-Glycoprotein Expression

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2020/4861719 ◽

2020 ◽

Vol 2020 ◽

pp. 1-8

Author(s):

Yali Liu ◽

Ling Zhang ◽

Shaofeng Wei ◽

Jinyang Cai ◽

Zhenzhong Zang ◽

...

Keyword(s):

Membrane Vesicles ◽

Pcr Analysis ◽

Mrna Levels ◽

Intestinal Epithelial ◽

Glycoprotein P ◽

P Glycoprotein ◽

Human Intestinal Epithelial Cells ◽

Pulsatilla Chinensis ◽

Dependent Transport ◽

Cytotoxic Studies

Five pulchinenosides (pulchinenoside B3, pulchinenoside BD, pulchinenoside B7, pulchinenoside B10, and pulchinenoside B11) isolated from Pulsatilla chinensis (Bge) Regel saponins extract exhibited strong antitumor activities but poor gastrointestinal absorption properties. The enteric induction of P-glycoprotein (P-gp) is understood to restrict the oral bioavailability of some pharmaceutical compounds and lead to adverse drug reactions. Therefore, the present investigation was intended to delineate the impacts of pulchinenosides on cellular P-gp function and expression using Sf9 membrane vesicles and LS180 cells as a surrogate of human intestinal epithelial cells. Preliminary cytotoxic studies showed that 10 μM was an acceptable concentration for cytotoxicity and antiproliferation studies for all pulchinenosides using the alamarBlue assay. The cell cycle of LS180 cells detected by flow cytometry was not significantly influenced after 48 hours of coincubation with 10 μM of pulchinenosides. In the presence of pulchinenosides, the ATP-dependent transport of N-methyl-quinidine mediated by P-glycoprotein was stimulated significantly. The upregulation of P-glycoprotein and mRNA levels was found by Western blot and real-time PCR analysis in LS180 cells. Parallel changes indicate that all pulchinenosides are exposed to pulchinenosides-mediated transcriptional regulation. In conclusion, pulchinenosides could induce P-glycoprotein expression and directly increase its functional activity.

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