RNAi of Grp78 may disturb the fusion of ICR mouse palate cultured in vitro

2017 ◽  
Vol 37 (2) ◽  
pp. 196-204 ◽  
Author(s):  
C Ren ◽  
X Li ◽  
X Li ◽  
Y Xie ◽  
H Fu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Blue Staining ◽  
Alcian Blue Staining ◽  
Unfolded Protein ◽  
All Trans Retinoic Acid ◽  
Icr Mouse ◽  
Activating Transcription Factor ◽  
The Unfolded Protein Response ◽  
Sirna Group

RNA interference (RNAi) is a powerful tool to silence or minimize gene expression, and palate culture in vitro is an important technique for study of the palate development. Our previous study demonstrated that the gene expression of glucose-regulated protein-78 (Grp78) was downregulation in the all-trans retinoic acid-induced mouse models of cleft palate (CP) during embryogenesis. To find the role of Grp78, the small interfering RNA (siRNA) of this gene carried by fluorescent vector was injected with a microinjector, through which about 30 pmol siRNA was injected into the Institute of Cancer Research (ICR) mouse palate explants. After 6, 12, 24, 48, and 72 h, these palate explants were removed from culture to observe their fluorescent and Alcian blue-staining phenotypes, and the expression of the unfolded protein response (UPR) key members (Grp78, Inositol-responsive enzyme 1, protein kinase RNA-like endoplasmic reticulum kinase, activating transcription factor-6 and X-box binding protein-1) was measured. After cultured for 72 h, the partially or completely fused bilateral palates were observed in the control siRNA group, while CPs were found in the Grp78 siRNA group. In the Grp78 siRNA group, the relatively mRNA abundance of the key genes belonged to UPR at each time point was lower than that of the control siRNA group, and their protein expression also displayed the same change. By the system of RNAi strategies with mouse palate culture, we found the siRNA of Grp78 disturbed the fusion of mouse palate cultured in vitro.

scholarly journals ATF6 is required for efficient rhodopsin clearance and retinal homeostasis in the P23H rho retinitis pigmentosa mouse model

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Eun-Jin Lee ◽  
Priscilla Chan ◽  
Leon Chea ◽  
Kyle Kim ◽  
Randal J. Kaufman ◽  
...  
Keyword(s):  
Retinitis Pigmentosa ◽  
Retinal Degeneration ◽  
Mrna Levels ◽  
Photoreceptor Layer ◽  
Protein Levels ◽  
Unfolded Protein ◽  
Activating Transcription Factor ◽  
The Unfolded Protein Response ◽  
Protein Response

AbstractRetinitis Pigmentosa (RP) is a blinding disease that arises from loss of rods and subsequently cones. The P23H rhodopsin knock-in (P23H-KI) mouse develops retinal degeneration that mirrors RP phenotype in patients carrying the orthologous variant. Previously, we found that the P23H rhodopsin protein was degraded in P23H-KI retinas, and the Unfolded Protein Response (UPR) promoted P23H rhodopsin degradation in heterologous cells in vitro. Here, we investigated the role of a UPR regulator gene, activating transcription factor 6 (Atf6), in rhodopsin protein homeostasis in heterozygous P23H rhodopsin (Rho+/P23H) mice. Significantly increased rhodopsin protein levels were found in Atf6−/−Rho+/P23H retinas compared to Atf6+/−Rho+/P23H retinas at early ages (~ P12), while rhodopsin mRNA levels were not different. The IRE1 pathway of the UPR was hyper-activated in young Atf6−/−Rho+/P23H retinas, and photoreceptor layer thickness was unchanged at this early age in Rho+/P23H mice lacking Atf6. By contrast, older Atf6−/−Rho+/P23H mice developed significantly increased retinal degeneration in comparison to Atf6+/−Rho+/P23H mice in all retinal layers, accompanied by reduced rhodopsin protein levels. Our findings demonstrate that Atf6 is required for efficient clearance of rhodopsin protein in rod photoreceptors expressing P23H rhodopsin, and that loss of Atf6 ultimately accelerates retinal degeneration in P23H-KI mice.

scholarly journals Investigating the physiological responses of Pacific white shrimp Litopenaeus vannamei to acute cold-stress

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e7381 ◽  
Author(s):  
Zhenlu Wang ◽  
Yuexin Qu ◽  
Xiaolei Zhuo ◽  
Junyi Li ◽  
Jixing Zou ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cold Stress ◽  
Litopenaeus Vannamei ◽  
Physiological Responses ◽  
Pacific White Shrimp ◽  
Basement Membranes ◽  
White Shrimp ◽  
Unfolded Protein ◽  
Activating Transcription Factor ◽  
The Unfolded Protein Response

Background Litopenaeus vannamei is one of the most important aquaculture shrimps in the world and low temperatures present a serious challenge to its survival, growth, and distribution. Methods To investigate their physiological responses during acute cold-stress, L. vannamei were treated under acute cooling conditions from 28 to 13 °C with a cooling rate of 2.5 °C/2 h and were maintained at 13 °C for 12 h. Plasma metabolite concentrations, histological changes, and relative gene expression related to the unfolded protein response (UPR) pathway and apoptosis in the hepatopancreas and the hemocytes of L. vannamei were investigated. Results The results revealed that the concentrations of triglycerides, total cholesterol, and total protein in plasma reached their peaks at 23 °C, and then decreased to their minimum values at 13 °C for 12 h. The activity of alkaline phosphatase in the plasma decreased to its lowest level while the activity of alanine aminotransferase increased to its highest level at 13 °C for 12 h. The hepatic tubules became necrotic and the basement membranes were ruptured at 13 °C for 12 h. The gene expression related to UPR and apoptosis in the hepatopancreas and hemocytes was significantly altered by the decrease in the temperature. Discussion The results revealed that acute cold-stress caused histological damage in the hepatopancreas of L. vannamei, reducing its immunity. The three UPR pathways were involved in the process of acute cold-stress and the response of activating transcription factor 6 to UPR may be faster and more directthan the IRE1 and PERK pathways.

scholarly journals The Effects of Bergamot Polyphenolic Fraction, Cynara cardunculus, and Olea europea L. Extract on Doxorubicin-Induced Cardiotoxicity

Nutrients ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 2158
Author(s):  
Jessica Maiuolo ◽  
Irene Bava ◽  
Cristina Carresi ◽  
Micaela Gliozzi ◽  
Vincenzo Musolino ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Apoptotic Cell ◽  
Natural Compounds ◽  
Cynara Cardunculus ◽  
Unfolded Protein ◽  
Wide Range ◽  
Vitro Model ◽  
The Unfolded Protein Response ◽  
Protein Response

Doxorubicin is an anthracycline that is commonly used as a chemotherapy drug due to its cytotoxic effects. The clinical use of doxorubicin is limited due to its known cardiotoxic effects. Treatment with anthracyclines causes heart failure in 15–17% of patients, resulting in mitochondrial dysfunction, the accumulation of reactive oxygen species, intracellular calcium dysregulation, the deterioration of the cardiomyocyte structure, and apoptotic cell death. Polyphenols have a wide range of beneficial properties, and particular importance is given to Bergamot Polyphenolic Fraction; Oleuropein, one of the main polyphenolic compounds of olive oil; and Cynara cardunculus extract. These natural compounds have particular beneficial characteristics, owing to their high polyphenol contents. Among these, their antioxidant and antoproliferative properties are the most important. The aim of this paper was to investigate the effects of these three plant derivatives using an in vitro model of cardiotoxicity induced by the treatment of rat embryonic cardiomyoblasts (H9c2) with doxorubicin. The biological mechanisms involved and the crosstalk existing between the mitochondria and the endoplasmic reticulum were examined. Bergamot Polyphenolic Fraction, Oleuropein, and Cynara cardunculus extract were able to decrease the damage induced by exposure to doxorubicin. In particular, these natural compounds were found to reduce cell mortality and oxidative damage, increase the lipid content, and decrease the concentration of calcium ions that escaped from the endoplasmic reticulum. In addition, the direct involvement of this cellular organelle was demonstrated by silencing the ATF6 arm of the Unfolded Protein Response, which was activated after treatment with doxorubicin.

scholarly journals Activating Transcription Factor 4 Is Translationally Regulated by Hypoxic Stress

2004 ◽  
Vol 24 (17) ◽  
pp. 7469-7482 ◽  
Author(s):  
Jaime D. Blais ◽  
Vasilisa Filipenko ◽  
Meixia Bi ◽  
Heather P. Harding ◽  
David Ron ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Microarray Analysis ◽  
Initiation Factor ◽  
Hypoxic Stress ◽  
Unfolded Protein ◽  
Activating Transcription Factor 4 ◽  
Activating Transcription Factor ◽  
The Unfolded Protein Response ◽  
Protein Response ◽  
Transcription Factor 4

ABSTRACT Hypoxic stress results in a rapid and sustained inhibition of protein synthesis that is at least partially mediated by eukaryotic initiation factor 2α (eIF2α) phosphorylation by the endoplasmic reticulum (ER) kinase PERK. Here we show through microarray analysis of polysome-bound RNA in aerobic and hypoxic HeLa cells that a subset of transcripts are preferentially translated during hypoxia, including activating transcription factor 4 (ATF4), an important mediator of the unfolded protein response. Changes in mRNA translation during the unfolded protein response are mediated by PERK phosphorylation of the translation initiation factor eIF2α at Ser-51. Similarly, PERK is activated and is responsible for translational regulation under hypoxic conditions, while inducing the translation of ATF4. The overexpression of a C-terminal fragment of GADD34 that constitutively dephosphorylates eIF2α was able to attenuate the phosphorylation of eIF2α and severely inhibit the induction of ATF4 in response to hypoxic stress. These studies demonstrate the essential role of ATF4 in the response to hypoxic stress, define the pathway for its induction, and reveal that GADD34, a target of ATF4 activation, negatively regulates the eIF2α-mediated inhibition of translation. Taken with the concomitant induction of additional ER-resident proteins identified by our microarray analysis, this study suggests an important integrated response between ER signaling and the cellular adaptation to hypoxic stress.

scholarly journals Transcriptome Analysis of Recombinant Protein Secretion by Aspergillus nidulans and the Unfolded-Protein Response In Vivo

2005 ◽  
Vol 71 (5) ◽  
pp. 2737-2747 ◽  
Author(s):  
Andrew H. Sims ◽  
Manda E. Gent ◽  
Karin Lanthaler ◽  
Nigel S. Dunn-Coleman ◽  
Stephen G. Oliver ◽  
...  
Keyword(s):  
Gene Expression ◽  
Recombinant Protein ◽  
Unfolded Protein Response ◽  
Aspergillus Nidulans ◽  
Filamentous Fungi ◽  
Protein Secretion ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Protein Response

ABSTRACT Filamentous fungi have a high capacity for producing large amounts of secreted proteins, a property that has been exploited for commercial production of recombinant proteins. However, the secretory pathway, which is key to the production of extracellular proteins, is rather poorly characterized in filamentous fungi compared to yeast. We report the effects of recombinant protein secretion on gene expression levels in Aspergillus nidulans by directly comparing a bovine chymosin-producing strain with its parental wild-type strain in continuous culture by using expressed sequence tag microarrays. This approach demonstrated more subtle and specific changes in gene expression than those observed when mimicking the effects of protein overproduction by using a secretion blocker. The impact of overexpressing a secreted recombinant protein more closely resembles the unfolded-protein response in vivo.

scholarly journals Η χρήση του πεπτιδίου Ec της ισόμορφης IGF-1Ec στην επισκευή χόνδρινων βλαβών

2018 ◽  
Author(s):  
Νικόλαος Αρμακόλας
Keyword(s):  
Polymerase Chain Reaction ◽  
Trypan Blue ◽  
Quantitative Polymerase Chain Reaction ◽  
Blue Staining ◽  
Alcian Blue Staining ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Tgf Β1 ◽  
Invasion Assays

Το πεπτίδιο Ec (PEc) του IGF-1Ec (IGF-1Ec) επάγει την κινητοποίηση των ανθρωπίνων μεσεγχυματικών βλαστικών κυττάρων (hMSC) και ενεργοποιεί την εξωκυτταρική κινάση 1 και 2 (ERK 1/2) διαφόρων κυττάρων. Σκοπός της παρούσας μελέτης ήταν η διερεύνηση της επιδρασης του PEc στην κινητοποίηση και τη διαφοροποίηση των hMSCs, καθώς και η δυνατότητα εφαρμογής του σε συνδυασμό με τον TGF-β1 (TGF-β1) στην επιδιόρθωση του αρθρικού χόνδρου. Τα αποτελέσματα της εξωγενούς χορήγησης του ΡΕc και του ΤGF-β1, ξεχωριστά και σε συνδυασμό, σε hMSCs εκτιμήθηκαν χρησιμοποιώντας trypan blue assay, reverse transcription-quantitative polymerase chain reaction, western blot analysis, Alcian blue staining, wound healing assays και migration/invasion assays. Προσδιορίστηκε ότι το PEc εμπλέκεται στη διαδικασία διαφοροποίησης των hMSCs προς υαλώδη χόνδρο. Η χορήγηση PEc ή / και TGF-β1 σε hMSCs έδειξε συγκρίσιμη εναπόθεση χονδρικής θεμέλειας ουσίας. Ακόμα, η χορήγηση του ΡΕc σε συνδυασμό με τον ΤGF-β1 συσχετίστηκε με μια σημαντική αύξηση στην κινητοποίηση των hMSC σε σύγκριση με την χορήγηση μόνο του TGF-β1 ή του ΡEc (Ρ <0,05). Επομένως, το ΡΕc φαίνεται να διευκολύνει in vitro την κινητοποίηση των hMSC και την διαφοροποίηση τους προς χονδροκύτταρα, ενισχύοντας το ρόλο του ΤGF-β1.

scholarly journals Enforced dimerization between XBP1s and ATF6f enhances the protective effects of the unfolded protein response (UPR) in models of neurodegeneration

2020 ◽  
Author(s):  
René L. Vidal ◽  
Denisse Sepulveda ◽  
Paulina Troncoso-Escudero ◽  
Paula Garcia-Huerta ◽  
Constanza Gonzalez ◽  
...  
Keyword(s):  
Gene Expression ◽  
Unfolded Protein Response ◽  
Target Genes ◽  
Cell Function ◽  
Alpha Synuclein ◽  
Protective Effects ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Protein Response

AbstractAlteration to endoplasmic reticulum (ER) proteostasis is observed on a variety of neurodegenerative diseases associated with abnormal protein aggregation. Activation of the unfolded protein response (UPR) enables an adaptive reaction to recover ER proteostasis and cell function. The UPR is initiated by specialized stress sensors that engage gene expression programs through the concerted action of the transcription factors ATF4, ATF6f, and XBP1s. Although UPR signaling is generally studied as unique linear signaling branches, correlative evidence suggests that ATF6f and XBP1s may physically interact to regulate a subset of UPR-target genes. Here, we designed an ATF6f-XBP1s fusion protein termed UPRplus that behaves as a heterodimer in terms of its selective transcriptional activity. Cell-based studies demonstrated that UPRplus has stronger an effect in reducing the abnormal aggregation of mutant huntingtin and alpha-synuclein when compared to XBP1s or ATF6 alone. We developed a gene transfer approach to deliver UPRplus into the brain using adeno-associated viruses (AAVs) and demonstrated potent neuroprotection in vivo in preclinical models of Parkinson’s and Huntington’s disease. These results support the concept where directing UPR-mediated gene expression toward specific adaptive programs may serve as a possible strategy to optimize the beneficial effects of the pathway in different disease conditions.

scholarly journals IreA controls endoplasmic reticulum stress-induced autophagy and survival through homeostasis recovery

2018 ◽  
pp. MCB.00054-18 ◽  
Author(s):  
Eunice Domínguez-Martín ◽  
Laura Ongay-Larios ◽  
Laura Kawasaki ◽  
Olivier Vincent ◽  
Gerardo Coello ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Cell Survival ◽  
Eukaryotic Cell ◽  
Functional Link ◽  
Unfolded Protein ◽  
Transcriptional Changes ◽  
The Unfolded Protein Response ◽  
Protein Response

The Unfolded Protein Response (UPR) is an adaptive pathway that restores cellular homeostasis after endoplasmic reticulum (ER) stress. The ER-resident kinase/ribonuclease Ire1 is the only UPR sensor conserved during evolution. Autophagy, a lysosomal degradative pathway, also contributes to the recovery of cell homeostasis after ER-stress but the interplay between these two pathways is still poorly understood. We describe the Dictyostelium discoideum ER-stress response and characterize its single bonafide Ire1 orthologue, IreA. We found that tunicamycin (TN) triggers a gene-expression reprogramming that increases the protein folding capacity of the ER and alleviates ER protein load. Further, IreA is required for cell-survival after TN-induced ER-stress and is responsible for nearly 40% of the transcriptional changes induced by TN. The response of Dictyostelium cells to ER-stress involves the combined activation of an IreA-dependent gene expression program and the autophagy pathway. These two pathways are independently activated in response to ER-stress but, interestingly, autophagy requires IreA at a later stage for proper autophagosome formation. We propose that unresolved ER-stress in cells lacking IreA causes structural alterations of the ER, leading to a late-stage blockade of autophagy clearance. This unexpected functional link may critically affect eukaryotic cell survival under ER-stress.

scholarly journals USP19 deubiquitinating enzyme inhibits muscle cell differentiation by suppressing unfolded-protein response signaling

2015 ◽  
Vol 26 (5) ◽  
pp. 913-923 ◽  
Author(s):  
Benjamin Wiles ◽  
Miao Miao ◽  
Erin Coyne ◽  
Louise Larose ◽  
Andrey V. Cybulsky ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Unfolded Protein Response ◽  
Muscle Cell ◽  
Deubiquitinating Enzyme ◽  
Muscle Cell Differentiation ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Protein Response

USP19 deubiquitinating enzyme has two isoforms, cytoplasmic and endoplasmic reticulum (ER) localized. The ER-localized isoform specifically suppresses muscle cell differentiation in vitro and appears to do so by inhibiting the unfolded-protein response that occurs during such differentiation. In vivo, loss of USP19 promotes muscle regeneration following injury.

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