The Protein Expression Level of a Heterogeneous Gene Inserted in LIPI-1 of the Listeria ivanovii Genome Relies on Its Insertion Orientation

2017 ◽  
Vol 27 (5) ◽  
pp. 269-276
Author(s):  
Sijing Liu ◽  
Mingjuan Jiang ◽  
Lin Su ◽  
Tian Tang ◽  
Xiang Zhang ◽  
...  
Keyword(s):  
Protein Expression ◽  
Bacterial Genome ◽  
Expression Level ◽  
Foreign Gene ◽  
Protein Expression Level ◽  
B Genome ◽  
Listeria Ivanovii ◽  
Cellular Immune

Due to its capability to multiply in either phagocytic or nonphagocytic cells, and to subsequently elicit a robust cellular immune response, <i>Listeria ivanovii</i> (LI) is thought to be feasible for developing bacteria-based live attenuated vaccines. We previously generated several recombinant LI strains expressing <i>Mycobacterium tuberculosis</i> antigens. Since the expression level of heterogeneous protein was sometimes very low, we attempted to elucidate the principle of heterogeneous protein expression in such recombinant LI strains. In this study, we inserted the <i>M. tuberculosis </i>antigen gene <i>Rv0129c </i>into LI strains at the same site as the genome but with a different insertion orientation. RT-qPCR and Western blot showed that when the insertion orientation of the heterogeneous gene was opposite to the <i>LIorfXYZ </i>gene in the<i> Listeria </i>pathogenicity island 1 in the bacterial genome, the heterogeneous gene could be transcribed well but the protein expression level seemed limited, both in vitro and in vivo. When inserted at an orientation consistent with <i>LIorfXYZ</i> at the same site in the genome, the expected 43-kD protein was observed in vitro as well as in a mouse model. Bacterial virulence was found to have decreased after recombination. This work confirms that the protein expression level of the heterogenous gene in such genome-recombinant LI-based vaccines is related to its inserted orientation in the bacterial genome, and a foreign gene inserted at this position of LIPI-1 will abolish <i>Listeria</i> virulence without affecting its growth.

scholarly journals Phosphorylation at Serines 157 and 161 Is Necessary for Preserving Cardiac Expression Level and Functions of Sarcomeric Z-Disc Protein Telethonin

2021 ◽  
Vol 12 ◽  
Author(s):  
Hannah R. Lewis ◽  
Seda Eminaga ◽  
Mathias Gautel ◽  
Metin Avkiran
Keyword(s):  
Protein Expression ◽  
Gene Dosage ◽  
Systolic Dysfunction ◽  
Ubiquitin Proteasome System ◽  
Expression Level ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Adrenergic Stimulation ◽  
Protein Expression Level

Aims: In cardiac myocytes, the sarcomeric Z-disc protein telethonin is constitutively bis-phosphorylated at C-terminal residues S157 and S161; however, the functional significance of this phosphorylation is not known. We sought to assess the significance of telethonin phosphorylation in vivo, using a novel knock-in (KI) mouse model generated to express non-phosphorylatable telethonin (TcapS157/161A).Methods and Results:TcapS157/161A and wild-type (WT) littermates were characterized by echocardiography at baseline and after sustained β-adrenergic stimulation via isoprenaline infusion. Heart tissues were collected for gravimetric, biochemical, and histological analyses. At baseline, TcapS157/161A mice did not show any variances in cardiac structure or function compared with WT littermates and mutant telethonin remained localized to the Z-disc. Ablation of telethonin phosphorylation sites resulted in a gene-dosage dependent decrease in the cardiac telethonin protein expression level in mice carrying the S157/161A alleles, without any alteration in telethonin mRNA levels. The proteasome inhibitor MG132 significantly increased the expression level of S157/161A telethonin protein in myocytes from TcapS157/161A mice, but not telethonin protein in myocytes from WT mice, indicating a role for the ubiquitin–proteasome system in the regulation of telethonin protein expression level. TcapS157/161A mice challenged with sustained β-adrenergic stimulation via isoprenaline infusion developed cardiac hypertrophy accompanied by mild systolic dysfunction. Furthermore, the telethonin protein expression level was significantly increased in WT mice following isoprenaline stimulation but this response was blunted in TcapS157/161A mice.Conclusion: Overall, these data reveal that telethonin protein turnover in vivo is regulated in a novel phosphorylation-dependent manner and suggest that C-terminal phosphorylation may protect telethonin against proteasomal degradation and preserve cardiac function during hemodynamic stress. Given that human telethonin C-terminal mutations have been associated with cardiac and skeletal myopathies, further research on their potential impact on phosphorylation-dependent regulation of telethonin protein expression could provide valuable mechanistic insight into those myopathies.

Relationship Between Post-Translational Modification of CD20 Protein and the Responsiveness to Rituximab Treatment

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2667-2667 ◽  
Author(s):  
Takumi Sugimoto ◽  
Akihiro Tomita ◽  
Kazuyuki Shimada ◽  
Junji Hiraga ◽  
Hitoshi Kiyoi ◽  
...  
Keyword(s):  
Protein Expression ◽  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Expression Level ◽  
Adcc Activity ◽  
Post Translational Modification ◽  
Protein Expression Level ◽  
Down Regulation

Abstract Rituximab is a murine/human chimeric-anti-CD20 monoclonal antibody that has become a key molecular-targeting drug for CD20-positive B-cell lymphomas. Although combination chemotherapy with rituximab has provided remarkably favorable results for CD20-positive B-cell lymphoma patients, acquired resistance to rituximab has become a considerable problem. Several mechanisms of resistance have been predicted, but the clinical significance of those mechanisms has remained unclear. Previously, at the last ASH Meeting, we showed that down-regulation of CD20 protein expression after using rituximab-containing chemotherapy is one of the critical mechanisms of rituximab resistance, and some epigenetic mechanisms, in part, were related to the aberrant down-regulation of MS4A1 (CD20) gene transcription. On the other hand, we have also found that some patients show resistance to rituximab even in the presence of CD20 protein expression. With this backgrounds in mind, we here investigated he relationship between CD20 protein expression condition and the responsiveness to treatment with rituximab. First, using B-cell lymphoma cell lines and primary lymphoma cells obtained from B-cell lymphoma patients, the CD20 protein expression condition was confirmed by immuno-histochemistry, flow cytometry (FCM), and immunoblotting (IB). In IB analysis, two different sizes of CD20 protein, upper (~37 kDa) and lower (~35 kDa), were confirmed. In vitro phosphatase assay suggested that the upper band is a phosphorylated band of the wild-type CD20 protein. The intensity of those two bands was measured, and the upper/lower (U/L) ratio was calculated. Interestingly, the U/L ratio in diffuse large B-cell lymphoma (DLBCL) cells was significantly lower (range; 0.1 to 0.3) than that of grade 1 and 2 follicular lymphoma (FL) and B-chronic lymphocytic leukemia (B-CLL) cells (0.3 to 0.9 and 0.5 to 0.9, respectively). Next, we tried to analyze the rituximab-inducing complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) activities using in vitro chrome-releasing assay. Generally, the CDC/ADCC activity in the CD20(−) cells was significantly lower than that of CD20(+) rituximab-sensitive cells. In contrast, the CDC/ADCC activity in CD20(+) cells varied considerably, with the result that the protein expression level measured by FCM and IB was not always proportional to rituximab sensitivity. In one FL patient showing clinically rituximab resistance in spite of the CD20(+) phenotype, CDC/ADCC activity in vitro was also significantly lower than that of rituximab-sensitive control cells, and the U/L ratio was remarkably high (~1.7) compared to CD20(+) DLBCL and FL cells obtained from rituximab-sensitive patents. Our findings suggest that some kind of B-cell lymphoma, e.g. DLBCL, can be differently classified by CD20 protein expression pattern according to the U/L ratio, and the responsiveness to rituximab may not be determined only by the whole CD20 protein expression level. Furthermore, our data may also suggest a possibility that the responsiveness to rituximab is, in part, modulated by post-translational modification of CD20 protein in vivo.

scholarly journals Upregulation of ETV2 Expression Promotes Endothelial Differentiation of Human Dental Pulp Stem Cells

2021 ◽  
Vol 30 ◽  
pp. 096368972097873
Author(s):  
Jing Li ◽  
Youming Zhu ◽  
Na Li ◽  
Tao Wu ◽  
Xianyu Zheng ◽  
...  
Keyword(s):  
Protein Expression ◽  
Dental Pulp ◽  
Tube Formation ◽  
Endothelial Differentiation ◽  
Cell Source ◽  
Lentiviral Infection ◽  
Mrna And Protein Expression ◽  
Human Dental Pulp

The lack of vasculogenesis often hampers the survivability and integration of newly engineered tissue grafts within the host. Autologous endothelial cells (ECs) are an ideal cell source for neovascularization, but they are limited by their scarcity, lack of proliferative capacity, and donor site morbidity upon isolation. The objective of this study was to determine whether differentiation of human dental pulp stem cells (DPSCs) into the endothelial lineage can be enhanced by recombinant ETV2 overexpression. DPSCs were extracted from fresh dental pulp tissues. ETV2 overexpression in DPSCs was achieved by lentiviral infection and cellular morphological changes were evaluated. The mRNA and protein expression levels of endothelial-specific markers were assessed through quantitative real-time polymerase chain reaction, western blot, immunofluorescence staining, and flow cytometry. The tube formation assay and Matrigel plug assay were also performed to evaluate the angiogenic potential of the ETV2-transduced cells in vitro and in vivo, respectively. Additionally, proteomic analysis was performed to analyze global changes in protein expression following ETV2 overexpression. After lentiviral infection, ETV2-overexpressing DPSCs showed endothelial-like morphology. Compared with control DPSCs, significantly higher mRNA and protein expression levels of endothelial-specific genes, including CD31, VE-Cadherin, VEGFR1, and VEGFR2, were detected in ETV2-overexpressing DPSCs. Moreover, ETV2 overexpression enhanced capillary-like tube formation on Matrigel in vitro, as well as neovascularization in vivo. In addition, comparative proteomic profiling showed that ETV2 overexpression upregulated the expression of vascular endothelial growth factor (VEGF) receptors, which was indicative of increased VEGF signaling. Taken together, our results indicate that ETV2 overexpression significantly enhanced the endothelial differentiation of DPSCs. Thus, this study shows that DPSCs can be a promising candidate cell source for tissue engineering applications.

scholarly journals Honeysuckle Aqueous Extracts Induced let-7a Suppress EV71 Replication and Pathogenesis In Vitro and In Vivo and Is Predicted to Inhibit SARS-CoV-2

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 308
Author(s):  
Ying-Ray Lee ◽  
Chia-Ming Chang ◽  
Yuan-Chieh Yeh ◽  
Chi-Ying F. Huang ◽  
Feng-Mao Lin ◽  
...  
Keyword(s):  
Protein Expression ◽  
Western Blotting ◽  
Expression Profiles ◽  
Plaque Assay ◽  
Virus Titer ◽  
Enterovirus 71 ◽  
Luciferase Reporter ◽  
Pathogen Invasion

Honeysuckle (Lonicera japonica Thunb) is a traditional Chinese medicine (TCM) with an antipathogenic activity. MicroRNAs (miRNAs) are small non-coding RNA molecules that are ubiquitously expressed in cells. Endogenous miRNA may function as an innate response to block pathogen invasion. The miRNA expression profiles of both mice and humans after the ingestion of honeysuckle were obtained. Fifteen overexpressed miRNAs overlapped and were predicted to be capable of targeting three viruses: dengue virus (DENV), enterovirus 71 (EV71) and SARS-CoV-2. Among them, let-7a was examined to be capable of targeting the EV71 RNA genome by reporter assay and Western blotting. Moreover, honeysuckle-induced let-7a suppression of EV71 RNA and protein expression as well as viral replication were investigated both in vitro and in vivo. We demonstrated that let-7a targeted EV71 at the predicted sequences using luciferase reporter plasmids as well as two infectious replicons (pMP4-y-5 and pTOPO-4643). The suppression of EV71 replication and viral load was demonstrated in two cell lines by luciferase activity, RT-PCR, real-time PCR, Western blotting and plaque assay. Furthermore, EV71-infected suckling mice fed honeysuckle extract or inoculated with let-7a showed decreased clinical scores and a prolonged survival time accompanied with decreased viral RNA, protein expression and virus titer. The ingestion of honeysuckle attenuates EV71 replication and related pathogenesis partially through the upregulation of let-7a expression both in vitro and in vivo. Our previous report and the current findings imply that both honeysuckle and upregulated let-7a can execute a suppressive function against the replication of DENV and EV71. Taken together, this evidence indicates that honeysuckle can induce the expression of let-7a and that this miRNA as well as 11 other miRNAs have great potential to prevent and suppress EV71 replication.

scholarly journals Comprehensive Analysis of ESRRA in Endometrial Cancer

2021 ◽  
Vol 20 ◽  
pp. 153303382199208
Author(s):  
Shufang Wang ◽  
Xinlong Huo
Keyword(s):  
Endometrial Cancer ◽  
Protein Expression ◽  
Immune Cell ◽  
Enrichment Analysis ◽  
Gene Set Enrichment Analysis ◽  
The Cancer Genome Atlas ◽  
Expression Level ◽  
Operating Characteristics ◽  
Protein Expression Level ◽  
Normal Tissues

Background: Estrogen-related receptor alpha (ESRRA) was reported to play an important role in multiple biological processes of neoplastic diseases. The roles of ESRRA in endometrial cancer have not been fully investigated yet. Methods: Expression data and clinicopathological data of patients with uteri corpus endometrial carcinoma (UCEC) were obtained from The Cancer Genome Atlas (TCGA). Comprehensive bioinformatics analysis was performed, including receiver operating characteristics (ROC) curve analysis, Kaplan-Meier survival analysis, gene ontology (GO) enrichment analysis, and Gene Set Enrichment Analysis (GSEA). Immunohistochemistry was used to detect the protein expression level of ESRRA and CCK-8 assay was performed to evaluate the effect of ESRRA on the proliferation ability. Results: A total of 552 UCEC tissues and 35 normal tissues were obtained from the TCGA database. The mRNA and protein expression level of ESRRA was highly elevated in UCEC compared with normal tissues, and was closely associated with poor prognosis. ROC analysis indicated a very high diagnostic value of ESRRA for patients with UCEC. GO and GSEA functional analysis showed that ESRRA might be mainly involved in cellular metabolism processes, in turn, tumorigenesis and progression of UCEC. Knockdown of ESRRA inhibited the proliferation of UCEC cells in vitro. Further immune cell infiltration demonstrated that ESRRA enhanced the infiltration level of neutrophil cell and reduced that of T cell (CD4+ naïve), NK cell, and cancer associated fibroblast (CAF). The alteration of immune microenvironment will greatly help in developing immune checkpoint therapy for UCEC. Conclusions: Our study comprehensively analyzed the expression level, clinical value, and possible mechanisms of action of ESRRA in UCEC. These findings showed that ESRRA might be a potential diagnostic and therapeutic target.

scholarly journals The inhibitory effect of silencing CDCA3 on migration and proliferation in bladder urothelial carcinoma

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Dexin Shen ◽  
Yayun Fang ◽  
Fenfang Zhou ◽  
Zhao Deng ◽  
Kaiyu Qian ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Urothelial Carcinoma ◽  
Carcinoma Cells ◽  
Expression Level ◽  
Tumor Cell Growth ◽  
Migration Ability ◽  
Bladder Urothelial Carcinoma ◽  
Related Proteins

Abstract Background CDCA3 is an important component of the E3 ligase complex with SKP1 and CUL1, which could regulate the progress of cell mitosis. CDCA3 has been widely identified as a proto-oncogene in multiple human cancers, however, its role in promoting human bladder urothelial carcinoma has not been fully elucidated. Methods Bioinformatic methods were used to analyze the expression level of CDCA3 in human bladder urothelial carcinoma tissues and the relationship between its expression level and key clinical characteristics. In vitro studies were performed to validate the specific functions of CDCA3 in regulating cell proliferation, cell migration and cell cycle process. Alterations of related proteins was investigated by western blot assays. In vivo studies were constructed to validate whether silencing CDCA3 could inhibit the proliferation rate in mice model. Results Bioinformatic analysis revealed that CDCA3 was significantly up-regulated in bladder urothelial carcinoma samples and was related to key clinical characteristics, such as tumor grade and metastasis. Moreover, patients who had higher expression level of CDCA3 tend to show a shorter life span. In vitro studies revealed that silencing CDCA3 could impair the migration ability of tumor cells via down-regulating EMT-related proteins such as MMP9 and Vimentin and inhibit tumor cell growth via arresting cells in the G1 cell cycle phase through regulating cell cycle related proteins like p21. In vivo study confirmed that silencing CDCA3 could inhibit the proliferation of bladder urothelial carcinoma cells. Conclusions CDCA3 is an important oncogene that could strengthen the migration ability of bladder urothelial carcinoma cells and accelerate tumor cell growth via regulating cell cycle progress and is a potential biomarker of bladder urothelial carcinoma.

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