scholarly journals Development of an in vitro model and fluorescent protein expression system for the study of highly abortigenic Campylobacter jejuni

2014 ◽  
Author(s):  
Moria Ann Borys
Keyword(s):  
Protein Expression ◽  
Campylobacter Jejuni ◽  
In Vitro Model ◽  
Fluorescent Protein ◽  
Expression System ◽  
Protein Expression System ◽  
Vitro Model

scholarly journals MicroRNA-1297 suppressed the Akt/GSK3β signaling pathway and stimulated neural apoptosis in an in vivo sevoflurane exposure model

2021 ◽  
Vol 49 (4) ◽  
pp. 030006052098210
Author(s):  
Quan Wang ◽  
Jingcong Luo ◽  
Ruiqiang Sun ◽  
Jia Liu
Keyword(s):  
Protein Expression ◽  
Signaling Pathway ◽  
Neuronal Apoptosis ◽  
In Vitro Model ◽  
Nervous System Development ◽  
Exposure Model ◽  
Control Group ◽  
Pten Protein ◽  
Vitro Model

Objective Common inhalation anesthetics used for clinical anesthesia (such as sevoflurane) may induce nerve cell apoptosis during central nervous system development. Furthermore, anesthetics can produce cognitive impairments, such as learning and memory impairments, that continue into adulthood. However, the precise mechanism remains largely undefined. We aimed to determine the function of microRNA-1297 (miR-1297) in sevoflurane-induced neurotoxicity. Methods Reverse transcription-polymerase chain reaction assays were used to analyze miR-1297 expression in sevoflurane-exposed mice. MTT and lactate dehydrogenase (LDH) assays were used to measure cell growth, and neuronal apoptosis was analyzed using flow cytometry. Western blot analyses were used to measure PTEN, PI3K, Akt, and GSK3β protein expression. Results In sevoflurane-exposed mice, miR-1297 expression was up-regulated compared with the control group. MiR-1297 up-regulation led to neuronal apoptosis, inhibition of cell proliferation, and increased LDH activity in the in vitro model of sevoflurane exposure. MiR-1297 up-regulation also suppressed the Akt/GSK3β signaling pathway and induced PTEN protein expression in the in vitro model. PTEN inhibition (VO-Ohpic trihydrate) reduced PTEN protein expression and decreased the effects of miR-1297 down-regulation on neuronal apoptosis in the in vitro model. Conclusion Collectively, the results indicated that miR-1297 stimulates sevoflurane-induced neurotoxicity via the Akt/GSK3β signaling pathway by regulating PTEN expression.

scholarly journals Improving Baculovirus Infectivity by Efficiently Embedding Enhancing Factors into Occlusion Bodies

2017 ◽  
Vol 83 (14) ◽  
Author(s):  
Shili Yang ◽  
Lijuan Zhao ◽  
Ruipeng Ma ◽  
Wei Fang ◽  
Jia Hu ◽  
...  
Keyword(s):  
Protein Expression ◽  
Fluorescent Protein ◽  
Expression System ◽  
Agrotis Segetum ◽  
Foreign Protein ◽  
Content Type ◽  
Occlusion Bodies ◽  
Foreign Proteins ◽  
Novel Strategy

ABSTRACT The relatively low infectivity of baculoviruses to their host larvae limits their use as insecticidal agents on a larger scale. In the present study, a novel strategy was developed to efficiently embed foreign proteins into Autographa californica multiple nucleopolyhedrovirus (AcMNPV) occlusion bodies (OBs) to achieve stable expression of foreign proteins and to improve viral infectivity. A recombinant AcMNPV bacmid was constructed by expressing the 150-amino-acid (aa) N-terminal segment of polyhedrin under the control of the p10 promoter and the remaining C-terminal 95-aa segment under the control of the polyhedrin promoter. The recombinant virus formed OBs in Spodoptera frugiperda 9 cells, in which the occlusion-derived viruses were embedded in a manner similar to that for wild-type AcMNPV. Next, the 95-aa polyhedrin C terminus was fused to enhanced green fluorescent protein, and the recombinant AcMNPV formed fluorescent green OBs and was stably passaged in vitro and in vivo. The AcMNPV recombinants were further modified by fusing truncated Agrotis segetum granulovirus enhancin or truncated Cydia pomonella granulovirus ORF13 (GP37) to the C-terminal 95 aa of polyhedrin, and both recombinants were able to form normal OBs. Bioactivity assays indicated that the median lethal concentrations of these two AcMNPV recombinants were 3- to 5-fold lower than that of the control virus. These results suggest that embedding enhancing factors in baculovirus OBs by use of this novel technique may promote efficient and stable foreign protein expression and significantly improve baculovirus infectivity. IMPORTANCE Baculoviruses have been used as bioinsecticides for over 40 years, but their relatively low infectivity to their host larvae limits their use on a larger scale. It has been reported that it is possible to improve baculovirus infectivity by packaging enhancing factors within baculovirus occlusion bodies (OBs); however, so far, the packaging efficiency has been low. In this article, we describe a novel strategy for efficiently embedding foreign proteins into AcMNPV OBs by expressing N- and C-terminal (dimidiate) polyhedrin fragments (150 and 95 amino acids, respectively) as fusions to foreign proteins under the control of the p10 and polyhedrin promoters, respectively. When this strategy was used to embed an enhancing factor (enhancin or GP37) into the baculovirus OBs, 3- to 5-fold increases in baculoviral infectivity were observed. This novel strategy has the potential to create an efficient protein expression system and a highly efficient virus-based system for insecticide production in the future.

Identity of the renin cell is mediated by cAMP and chromatin remodeling: an in vitro model for studying cell recruitment and plasticity

2008 ◽  
Vol 294 (2) ◽  
pp. H699-H707 ◽  
Author(s):  
Ellen Steward Pentz ◽  
Maria Luisa S. Sequeira Lopez ◽  
Magali Cordaillat ◽  
R. Ariel Gomez
Keyword(s):  
Chromatin Remodeling ◽  
In Vitro Model ◽  
Molecular Mechanisms ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Histone H4 Acetylation ◽  
Renin Gene ◽  
Vitro Model

The renin-angiotensin system (RAS) regulates blood pressure and fluid-electrolyte homeostasis. A key step in the RAS cascade is the regulation of renin synthesis and release by the kidney. We and others have shown that a major mechanism to control renin availability is the regulation of the number of cells capable of making renin. The kidney possesses a pool of cells, mainly in its vasculature but also in the glomeruli, capable of switching from smooth muscle to endocrine renin-producing cells when homeostasis is threatened. The molecular mechanisms governing the ability of these cells to turn the renin phenotype on and off have been very difficult to study in vivo. We, therefore, developed an in vitro model in which cells of the renin lineage are labeled with cyan fluorescent protein and cells actively making renin mRNA are labeled with yellow fluorescent protein. The model allowed us to determine that it is possible to culture cells of the renin lineage for numerous passages and that the memory to express the renin gene is maintained in culture and can be reenacted by cAMP and chromatin remodeling (histone H4 acetylation) at the cAMP-responsive element in the renin gene.

scholarly journals Expression of Recombinant Fusion Protein of Newcastle Disease Virus from Escherichia coli Plasmid Clone C-2a by In-vitro Cell-free Protein Expression System

2020 ◽  
Vol 20 ◽  
pp. 04004
Author(s):  
Ahmad Pandu Satria Wiratama ◽  
Aris Haryanto
Keyword(s):  
Protein Expression ◽  
Newcastle Disease ◽  
Recombinant Plasmid ◽  
Expression System ◽  
Free Protein ◽  
F Protein ◽  
E Coli ◽  
Protein Expression System ◽  
Sds Page

Newcastle Disease Virus (NDV) is an infectious disease that infect many kinds of wild and domesticated birds. Infection of NDV become a massive problem for poultry industry around the world especially in Indonesia. Vaccination is an effort to prevent the infection of NDV in poultry. NDV vaccine that used in Indonesia is a conventional life vaccine from LaSota and B1 strains. These type of vaccine is 21%-23% genetically distinct with the virus that spread in the environment. The antibody protection provided by the vaccine is not effective. Therefore, vaccination with new local NDV strain is needed to prevent the NDV infection in Indonesia. The previously study research reported that the local isolate of NDV from Kulon Progo, Indonesia has been isolated. Fusion (F) protein encoding gene that has been inserted into pBT7-N-His expression p lasmid which isolated from clone C-2a of E. coli, then it was expressed by the Cell-free protein expression system. The aim of this study was to confirm whether clone C-2a of E.coli carrying a recombinant plasmid pBT7-N-His-Fusion NDV and to express a recombinant F protein of NDV in-vitro from expression plasmid by cell-free protein expression system. This work started by detection of recombinant plasmid pBT7-N-His-Fusion NDV by DNA plasmid extraction followed by agarose gel electrophoresis. The recombinant F protein was in-vitro expressed by cell-free protein expression kit. The expressed F protein of NDV then was visualized by SDS-PAGE and Westernblott to analyse the expression of NDV recombinant F protein. It confirmed that clone C-2a of E. coli contained plasmid pBT7-N-His (4.001 bp) inserted by recombinant F protein of NDV gene (642 bp). The visualisation of expressed recombinant F protein by SDS-PAGE and Westernblott showed the NDV recombinant F protein was a specific protein fragment with molecular weight of 25,6 kDa..

Proteomic Analysis Identifies HSP70 As a Novel Target Therapy to Polycythemia Vera

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2827-2827
Author(s):  
Miguel Gallardo ◽  
Marisol Fernandez ◽  
Alberto Paradela ◽  
Oscar Toldos ◽  
Rosa-Maria Garcia-Martin ◽  
...  
Keyword(s):  
Protein Expression ◽  
Polycythemia Vera ◽  
Healthy Subjects ◽  
In Vitro Model ◽  
2D Dige ◽  
Phenotypic Divergence ◽  
Healthy Donors ◽  
Proteomic Screening ◽  
Vitro Model

Abstract Abstract 2827 Background Proteomic screening is a useful tool to find new therapeutic targets. Genetic molecular studies are frequent in myeloproliferative neoplasms (MPN), however proteomic screening studies are limited. HSP70 is a Heat Shock protein related to apoptosis supression and erythroid differenciation through GATA-1. Additionally, other HSP, as HSP90. have been found as novel therapies in Polycythemia Vera (PV) and Essential thrombocythemia (ET) (Marubayashi et al, Journal of Clinical Investigation, 2010Oct). Aims To analyze the phenotypic divergence between PV and ET by a proteomic screening, and to validate these results by protein expression analysis and in vitro model of MPN, with the aim of identifying alternative routes for targeted therapy. Methods Seventy-one MPN diagnosed by WHO criteria were included in the study: 25 PV, 24 ET JAK2V617F, 12 ETJAK2 Wild Type (JAK2WT) negative and 11 Primary Myelofibrosis (PMF). Additionally 24 healthy subjects were used as controls. First of all, granulocytes from whole venous peripheral blood were isolated and the corresponding cytosolic protein fraction was extracted. Cytosolic proteomes of 10 PV, 10 ET and 10 healthy subjects were analyzed using 2D-DIGE gels followed by MALDI-TOFTOF mass spectrometry (MS) analysis of the spots of interest. Results were analyzed with DeCyder v7.0 and Mascot software. Secondly, bone marrow biopsies (BMB) of 55 MPN patients (11 PV, 11 ET JAK2V617F, 11 ET JAK2WT, 11 PMF and 11 healthy controls) were selected to perform immunohistochemistry (IHC) with anti-HSPA1A (HSP70), anti-SERPINB1 and anti-LTA4H. Afterwards, western blot of these three proteins was performed. Finally, an in vitro model of MPN was employed. Mononuclear cells from 4 PV, 4 ET and 3 healthy donors were extracted and seeded in Methocult with IL-3, SCF and EPO. A HSP70 inhibition assay was performed by the drug KNK437 at 100μM, 50μM and 10μM. Results were analyzed by BFU-E count, viability study by trypan blue and flow cytometry (FCM) employing anti-CD45, anti-CD41, anti-CD34, anti-CD71, and Annexin antibodies. Intracellular proteins including phospho-proteins p38, P-p38, MEK, P-MEK, STAT1, P-STAT1, AKT1 and P-AKT were studied by cytometric bead array multiplexed bead-based immunoassay (CBAs) technique. The Mann-Whitney non-parametrical statistical hypothesis test was used to assess the statistical significance of our results. Results 2D-DIGE analysis found 112 spots with statistically significant differences in protein expression between PV and ET samples. On the other hand, 241 spots showed differential expression between ET and healthy donors, and 229 between PV and healthy donors. We identified, by MS, 67 proteins differentially expressed between PV and ET, 20 between PV and controls and 6 between ET and controls. A large number of these proteins were metabolic and citoskeleton proteins, as Lactotranferrin, Enolase, Actin, etc. However, three spots were especially interesting according to our hypothesis: SERPINB1, LTA4H and HSPA1A (HSP70); the last, chaperone related with GATA-1 and erythroid differentiation. IHC showed over-expression of HSPA1A in granulocytes of PV (72% positives patients, 80% positive granulocytes) compared to ET (50% positive patients, 30% positive granulocytes). Finally, significant differences in inhibition of BFU-E growth and cell proliferation were found between cultures treated with HSP70 inhibitor, KNK437 (100μM and 50μM) versus cultures without treatment (P=0.029). FCM of BFU-E cultures pointed to a dramatic increase of erythroid apoptotic cells. CBAs analysis showed a decrease of P-STAT1 and P-MEK in cells of cultures under KNK437 treatment. Conclusions Our results suggest that HSP70 could play a key role in erythroid survival and differentiation through JAK-STAT and MAPK pathways. These molecules might contribute to their phenotypic divergence. Finally, HSP70 could be a new therapeutic target, and KNK437 a novel PV treatment. Disclosures: No relevant conflicts of interest to declare.

scholarly journals In vitro expression of the recombinant fusion protein of Newcastle disease virus from local Indonesian isolates by using a cell-free protein expression system

2020 ◽  
Vol 25 (2) ◽  
pp. 69
Author(s):  
Aris Haryanto ◽  
Hevi Wihadmadyatami ◽  
Nastiti Wijayanti
Keyword(s):  
Protein Expression ◽  
Newcastle Disease Virus ◽  
Restriction Endonuclease ◽  
Newcastle Disease ◽  
Expression System ◽  
Free Protein ◽  
F Protein ◽  
Protein Expression System ◽  
A Cell

The aim of this work was the in vitro expression of the recombinant fusion (F) protein of Newcastle disease virus (NDV).  The pBT7-N-His-Fusion-NDV expression plasmid which carries the recombinant F protein encoding gene from local Indonesian isolates, was prepared and transformed into E. coli BL21 (DE3). To detect bacterial colonies carrying the recombinant plasmid, a restriction endonuclease analysis was performed using the EcoRI restriction endonuclease. These results showed that the pBT-N-His-Fusion-NDV plasmid was successfully isolated with a size of 4.601 bp, and three recombinant plasmids carrying the gene coding for the recombinant F protein of NDV were obtained. Selected recombinant plasmids were then in vitro by using a cell-free protein expression system followed by visualization of the recombinant F protein on a 12% SDS-PAGE gel both by Coomassie Brilliant Blue staining and Western blotting. Recombinant F protein was successfully in vitro expressed by using a cell-free protein expression system as indicated by a specific single protein band with a molecular mass of 25.6 kDa.

scholarly journals MiRNA-221-5p suppressed the Th17/Treg ratio in asthma via RORγt/Foxp3 by targeting SOCS1

2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Yuanyuan Guan ◽  
Yuemei Ma ◽  
Yao Tang ◽  
Xiaolei Liu ◽  
Yan Zhao ◽  
...  
Keyword(s):  
Protein Expression ◽  
In Vitro Model ◽  
Treg Cells ◽  
Orphan Receptor ◽  
In Vivo Model ◽  
Cytokine Signaling ◽  
T Helper 17 ◽  
Vitro Model

Abstract Background This study was designed to investigate the mechanism and effects of miRNA-221-5p on the T-helper 17 (Th17)/T-regulatory (Treg) ratio in asthma. Methods BALB/c mice were intranasally challenged with 100 µg OVA on 14 and 21 day. Mice were rechallenged with 2.5% OVA-PBS on 22 and 28 day. Mice were sacrificed using on day 30 under 35 mg/kg pentobarbital sodium. PBMCs were induced vitro model of asthma using 500 ng of lipopolysaccharides (LPS) for 4 h. Results The expression of miRNA-221-5p was reduced in in vivo model, compared sham group. The vitro model of asthma treated with miRNA-221-5p mimic resulted in the reduction of IL-6, IL-17, IL-21 and IL-22 levels, and induction of IL-10, IL-35 and TGF-β levels. In addition, down-regulation of miRNA-221-5p induced the protein expression of suppressor of cytokine signaling 1 (SOCS1) and receptor-related orphan receptor-gamma-t (RORγt) and suppressed that of FOXP3 in in vitro model of asthma. Over-expression of miRNA-221-5p induced the protein expression of FOXP3, and suppressed that of SOCS1 and RORγt in in vitro model of asthma. The inhibition of SOCS1 or RORγt attenuated the effects of anti-miRNA-221-5p on Th17/Treg ratio in asthma. Conclusion miRNA-221-5p may play critical roles in driving the differentiation of Th17/Treg ratio via RORγt/Foxp3 by Targeting SOCS1, reduced the function of Th17 cells by directly inhibiting RORγt/SOCS1 and promoted the function of Treg cells via Foxp3/ SOCS1 in asthma.

scholarly journals Mechanism of glucocorticoid regulation of the intestinal tight junction barrier

2007 ◽  
Vol 292 (2) ◽  
pp. G590-G598 ◽  
Author(s):  
Michel A. Boivin ◽  
Dongmei Ye ◽  
John C. Kennedy ◽  
Rana Al-Sadi ◽  
Chris Shepela ◽  
...  
Keyword(s):  
Crohn’S Disease ◽  
Crohn's Disease ◽  
Protein Expression ◽  
Tight Junction ◽  
Barrier Function ◽  
In Vitro Model ◽  
Intestinal Epithelial ◽  
Tnf Α ◽  
Vitro Model

A defective intestinal epithelial tight junction (TJ) barrier has been proposed as an important pathogenic factor contributing to the intestinal inflammation of Crohn's disease. Glucocorticoids are first-line therapeutic agents for the treatment of moderate to severe Crohn's disease. Glucocorticoid treatment has been shown to induce retightening of the intestinal TJ barrier defect in Crohn's disease patients. However, the mechanisms that mediate the glucocorticoid therapeutic action on intestinal TJ barrier function remain unknown. The aim of this study was to elucidate the mechanism of glucocorticoid modulation of the intestinal epithelial TJ barrier using an in vitro model system. Filter-grown Caco-2 intestinal epithelial cells were used as an in vitro model to examine the effects of glucocorticoids on basal intestinal epithelial TJ barrier function and on TNF-α-induced disruption of the TJ barrier. Glucocorticoids (prednisolone and dexamethasone) did not have a significant effect on baseline Caco-2 TJ barrier function but prevented the TNF-α-induced increase in Caco-2 TJ permeability. The glucocorticoid protective effect against the TNF-α-induced increase in Caco-2 TJ permeability required activation of the glucocorticoid receptor (GR) complex. The activation of the GR complex resulted in GR complex binding to the glucocorticoid response element (GRE) site on DNA and activation of a GR-responsive promoter. Glucocorticoids inhibited the TNF-α-induced increase in myosin light chain kinase (MLCK) protein expression, a key process mediating the TNF-α increase in intestinal TJ permeability. The glucocorticoid inhibition of the TNF-α-induced increase in MLCK protein expression was due to the binding of the GR complex to a GRE binding site on the MLCK promoter region suppressing the TNF-α-induced activation. Glucocorticoids inhibit the TNF-α-induced increase in Caco-2 TJ permeability. The prednisolone protective action was mediated by binding of activated GR complex to the GRE site on the MLCK promoter, suppressing the TNF-α-induced increase in MLCK gene activity, protein expression, and subsequent opening of the intestinal TJ barrier.

CircRNA 001418 Promoted Cell Growth and Metastasis of Bladder Carcinoma via EphA2 by miR-1297

2020 ◽  
Vol 14 (1) ◽  
pp. 68-78 ◽  
Author(s):  
Guorui Peng ◽  
Hongxue Meng ◽  
Hongxin Pan ◽  
Wentao Wang
Keyword(s):  
Cell Proliferation ◽  
Cell Growth ◽  
Cytochrome C ◽  
Protein Expression ◽  
Bladder Carcinoma ◽  
In Vitro Model ◽  
Caspase 3 ◽  
Activity Levels ◽  
Vitro Model

Background: Cancer is one of the major causes of human deaths at present. It is the leading cause of deaths in developed countries. Moreover, Circular RNAs (circRNAs) have been discovered to play important roles in tumor genesis and development and are abnormally expressed in bladder cancer . Objective: The present study aims to investigate the anti-cancer effects of circ 001418 on bladder carcinoma and its possible mechanism. Methods: Quantitative PCR (qPCR) and gene chip were used to measure the circ 001418 expression. Cell proliferation and transfer, apoptosis and caspase-8 and caspase-3 activity levels were measured using MTT, Transwell assay, Flow cytometry. Caspase-3 and 9 activity levels, EphA2, cytochrome c and FADD protein expression were detected using Western blotting. Results: The expression of circ 001418 was increased in patients with bladder carcinoma. Over-expression of circ 001418 promoted cell proliferation and transfer, and reduced apoptosis in vitro model of bladder carcinoma. Down-regulation of Circ 001418 inhibited cell proliferation and transfer, and induced apoptosis in vitro model of bladder carcinoma. Meanwhile, Overexpression of circ 001418 induced EphA2 and cytochrome c protein expression, suppressed FADD protein expression in vitro model of bladder carcinoma by suppression of miR-1297. MiR-1297 reduced the pro-cancer effect of circ 001418 on apoptosis of bladder carcinoma. Conclusion: Results showed thatcircRNA 001418 promoted cell growth and metastasis of bladder carcinoma via EphA2 by miR-1297.

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