Examining rhodopsin retention in endoplasmic reticulum and intracellular localization in vitro and in vivo by using truncated rhodopsin fragments

2011 ◽  
Vol 112 (2) ◽  
pp. 520-530 ◽  
Author(s):  
Yuh-Fang Chen ◽  
I-Jong Wang ◽  
Luke L.K. Lin ◽  
Muh-Shy Chen
Keyword(s):  
Endoplasmic Reticulum ◽  
Intracellular Localization

scholarly journals Dehydrodiisoeugenol inhibits colorectal cancer growth by endoplasmic reticulum stress-induced autophagic pathways

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Changhong Li ◽  
Kui Zhang ◽  
Guangzhao Pan ◽  
Haoyan Ji ◽  
Chongyang Li ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Endoplasmic Reticulum ◽  
Cell Growth ◽  
Er Stress ◽  
Cancer Cells ◽  
Tumor Xenograft ◽  
Anticancer Activities ◽  
Colorectal Cancer Cells

Abstract Background Dehydrodiisoeugenol (DEH), a novel lignan component extracted from nutmeg, which is the seed of Myristica fragrans Houtt, displays noticeable anti-inflammatory and anti-allergic effects in digestive system diseases. However, the mechanism of its anticancer activity in gastrointestinal cancer remains to be investigated. Methods In this study, the anticancer effect of DEH on human colorectal cancer and its underlying mechanism were evaluated. Assays including MTT, EdU, Plate clone formation, Soft agar, Flow cytometry, Electron microscopy, Immunofluorescence and Western blotting were used in vitro. The CDX and PDX tumor xenograft models were used in vivo. Results Our findings indicated that treatment with DEH arrested the cell cycle of colorectal cancer cells at the G1/S phase, leading to significant inhibition in cell growth. Moreover, DEH induced strong cellular autophagy, which could be inhibited through autophagic inhibitors, with a rction in the DEH-induced inhibition of cell growth in colorectal cancer cells. Further analysis indicated that DEH also induced endoplasmic reticulum (ER) stress and subsequently stimulated autophagy through the activation of PERK/eIF2α and IRE1α/XBP-1 s/CHOP pathways. Knockdown of PERK or IRE1α significantly decreased DEH-induced autophagy and retrieved cell viability in cells treated with DEH. Furthermore, DEH also exhibited significant anticancer activities in the CDX- and PDX-models. Conclusions Collectively, our studies strongly suggest that DEH might be a potential anticancer agent against colorectal cancer by activating ER stress-induced inhibition of autophagy.

scholarly journals Overexpression of SERCA2a Alleviates Cardiac Microvascular Ischemic Injury by Suppressing Mfn2-Mediated ER/Mitochondrial Calcium Tethering

2021 ◽  
Vol 9 ◽  
Author(s):  
Feng Tian ◽  
Ying Zhang
Keyword(s):  
Endoplasmic Reticulum ◽  
Infarct Size ◽  
Myocardial Infarct ◽  
Ischemic Injury ◽  
Calcium Overload ◽  
Mitochondrial Calcium ◽  
Myocardial Infarct Size ◽  
Hypoxic Injury

Our previous research has shown that type-2a Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a) undergoes posttranscriptional oxidative modifications in cardiac microvascular endothelial cells (CMECs) in the context of excessive cardiac oxidative injury. However, whether SERCA2a inactivity induces cytosolic Ca2+ imbalance in mitochondrial homeostasis is far from clear. Mitofusin2 (Mfn2) is well known as an important protein involved in endoplasmic reticulum (ER)/mitochondrial Ca2+ tethering and the regulation of mitochondrial quality. Therefore, the aim of our study was to elucidate the specific mechanism of SERCA2a-mediated Ca2+ overload in the mitochondria via Mfn2 tethering and the survival rate of the heart under conditions of cardiac microvascular ischemic injury. In vitro, CMECs extracted from mice were subjected to 6 h of hypoxic injury to mimic ischemic heart injury. C57-WT and Mfn2KO mice were subjected to a 1 h ischemia procedure via ligation of the left anterior descending branch to establish an in vivo cardiac ischemic injury model. TTC staining, immunohistochemistry and echocardiography were used to assess the myocardial infarct size, microvascular damage, and heart function. In vitro, ischemic injury induced irreversible oxidative modification of SERCA2a, including sulfonylation at cysteine 674 and nitration at tyrosine 294/295, and inactivation of SERCA2a, which initiated calcium overload. In addition, ischemic injury-triggered [Ca2+]c overload and subsequent [Ca2+]m overload led to mPTP opening and ΔΨm dissipation compared with the control. Furthermore, ablation of Mfn2 alleviated SERCA2a-induced mitochondrial calcium overload and subsequent mito-apoptosis in the context of CMEC hypoxic injury. In vivo, compared with that in wild-type mice, the myocardial infarct size in Mfn2KO mice was significantly decreased. In addition, the findings revealed that Mfn2KO mice had better heart contractile function, decreased myocardial infarction indicators, and improved mitochondrial morphology. Taken together, the results of our study suggested that SERCA2a-dependent [Ca2+]c overload led to mitochondrial dysfunction and activation of Mfn2-mediated [Ca2+]m overload. Overexpression of SERCA2a or ablation of Mfn2 expression mitigated mitochondrial morphological and functional damage by modifying the SERCA2a/Ca2+-Mfn2 pathway. Overall, these pathways are promising therapeutic targets for acute cardiac microvascular ischemic injury.

scholarly journals Biochanin A Alleviates Cerebral Ischemia/Reperfusion Injury by Suppressing Endoplasmic Reticulum Stress-Induced Apoptosis and p38MAPK Signaling Pathway In Vivo and In Vitro

2021 ◽  
Vol 12 ◽  
Author(s):  
Min-min Guo ◽  
Sheng-biao Qu ◽  
Hui-ling Lu ◽  
Wen-bo Wang ◽  
Mu-Liang He ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
P38 Mapk ◽  
Ischemia Reperfusion ◽  
Biochanin A ◽  
Neurological Deficits ◽  
Mapk Phosphorylation ◽  
Cerebral Ischemia Reperfusion

We have previously shown that biochanin A exhibits neuroprotective properties in the context of cerebral ischemia/reperfusion (I/R) injury. The mechanistic basis for such properties, however, remains poorly understood. This study was therefore designed to explore the manner whereby biochanin A controls endoplasmic reticulum (ER) stress, apoptosis, and inflammation within fetal rat primary cortical neurons in response to oxygen-glucose deprivation/reoxygenation (OGD/R) injury, and in a rat model of middle cerebral artery occlusion and reperfusion (MCAO/R) injury. For the OGD/R in vitro model system, cells were evaluated after a 2 h OGD following a 24 h reoxygenation period, whereas in vivo neurological deficits were evaluated following 2 h of ischemia and 24 h of reperfusion. The expression of proteins associated with apoptosis, ER stress (ERS), and p38 MAPK phosphorylation was evaluated in these samples. Rats treated with biochanin A exhibited reduced neurological deficits relative to control rats following MCAO/R injury. Additionally, GRP78 and CHOP levels rose following I/R modeling both in vitro and in vivo, whereas biochanin A treatment was associated with reductions in CHOP levels but further increases in GRP78 levels. In addition, OGD/R or MCAO/R were associated with markedly enhanced p38 MAPK phosphorylation that was alleviated by biochanin A treatment. Similarly, OGD/R or MCAO/R injury resulted in increases in caspase-3, caspase-12, and Bax levels as well as decreases in Bcl-2 levels, whereas biochanin A treatment was sufficient to reverse these phenotypes. Together, these findings thus demonstrate that biochanin A can alleviate cerebral I/R-induced damage at least in part via suppressing apoptosis, ER stress, and p38 MAPK signaling, thereby serving as a potent neuroprotective agent.

scholarly journals AN ANALYSIS OF COLLAGEN SECRETION BY ESTABLISHED MOUSE FIBROBLAST LINES

1964 ◽  
Vol 22 (1) ◽  
pp. 227-258 ◽  
Author(s):  
Burton Goldberg ◽  
Howard Green
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Mouse Fibroblast ◽  
In Vivo Studies ◽  
In Vitro Synthesis ◽  
Collagen Secretion ◽  
Golgi System ◽  
Alternative Theories

In vitro synthesis of collagen by established mouse fibroblast lines has been examined by electron microscopy. During rapid growth (log phase), when collagen could not be detected in the cultures, the cells lacked a well developed granular ergastoplasm and Golgi system. Upon cessation of growth (stationary phase), collagen accumulated in the cultures and the cells demonstrated highly developed granular and smooth ergastoplasm. Collagen appeared to be synthesized in the rough-surfaced endoplasmic reticulum and to be transported as a soluble protein to the cell surface by vesicular elements of the agranular ergastoplasm. Fusion of the limiting membranes of these vesicles with the cell membrane permitted the discharge of the soluble collagen into the extracellular space, where fibrils of two diameter distributions formed. The secretion of collagen is concluded to be of the merocrine type. Alternative theories of collagen secretion are discussed and the data for established lines compared with the results of other in vitro and in vivo studies of collagen fibrillogenesis.

An Electron-Microscope Study of the in vitro Transformation of Human Leucocytes

1967 ◽  
Vol 2 (3) ◽  
pp. 359-370
Author(s):  
J. A. CHAPMAN ◽  
M. W. ELVES ◽  
J. GOUGH
Keyword(s):  
Endoplasmic Reticulum ◽  
Electron Microscope ◽  
Messenger Rna ◽  
Human Peripheral Blood ◽  
Limited Extent ◽  
Single Particles ◽  
Blast Cells ◽  
Protein Secreting

Electron-microscope studies of cultured small lymphocytes from human peripheral blood transforming into larger blastoid cells in the presence of phytohaemagglutinin (PHA) show that the transformed cell possesses the preliminary stages of development of a protein-synthesizing system. The transformed blastoid cell has abundant ribosomes, although, in contrast with in vivo protein-secreting cells, many of these occur as single particles with only a small proportion Linked in polysomal clusters. Endoplasmic reticulum membranes occur to a very limited extent and with a marked paucity of attached ribosomal particles; the few attached particles are usually located in groups. Some endoplasmic reticulum membranes revealed degenerative changes in otherwise normal cells. A moderately well-developed Golgi apparatus was a characteristic feature of the cells. Apart from the relatively low proportion of polysomes, in vitro PHA-transformed blastoid cells are identical in fine structure to in vivo blast cells (otherwise known as immunoblasts, haemocytoblasts, etc.) occurring in the immune response. It is suggested that messenger-RNA production in PHA-stimulated transformed cells may be reduced and that this could explain the limited number of polysomes and the restricted development of the endoplasmic reticulum.

scholarly journals Biosynthesis and processing of ribophorins in the endoplasmic reticulum.

1984 ◽  
Vol 99 (3) ◽  
pp. 1076-1082 ◽  
Author(s):  
M G Rosenfeld ◽  
E E Marcantonio ◽  
J Hakimi ◽  
V M Ort ◽  
P H Atkinson ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Posttranslational Modifications ◽  
Rat Hepatocytes ◽  
Direct Analysis ◽  
In Vitro Translation ◽  
Endoplasmic Reticulum Membrane ◽  
Pituitary Cells ◽  
Amino Terminal

Ribophorins are two transmembrane glycoproteins characteristic of the rough endoplasmic reticulum, which are thought to be involved in the binding of ribosomes. Their biosynthesis was studied in vivo using lines of cultured rat hepatocytes (clone 9) and pituitary cells (GH 3.1) and in cell-free synthesis experiments. In vitro translation of mRNA extracted from free and bound polysomes of clone 9 cells demonstrated that ribophorins are made exclusively on bound polysomes. The primary translation products of ribophorin messengers obtained from cultured hepatocytes or from regenerating livers co-migrated with the respective mature proteins, but had slightly higher apparent molecular weights (2,000) than the unglycosylated forms immunoprecipitated from cells treated with tunicamycin. This indicates that ribophorins, in contrast to all other endoplasmic reticulum membrane proteins previously studied, contain transient amino-terminal insertion signals which are removed co-translationally. Kinetic and pulse-chase experiments with [35S]methionine and [3H]mannose demonstrated that ribophorins are not subjected to electrophoretically detectable posttranslational modifications, such as proteolytic cleavage or trimming and terminal glycosylation of oligosaccharide side chain(s). Direct analysis of the oligosaccharides of ribophorin l showed that they do not contain the terminal sugars characteristic of complex oligosaccharides and that they range in composition from Man8GlcNAc to Man5GlcNAc. These findings, as well as the observation that the mature proteins are sensitive to endoglycosidase H and insensitive to endoglycosidase D, are consistent with the notion that the biosynthetic pathway of the ribophorins does not require a stage of passage through the Golgi apparatus.

scholarly journals Secretion ofSaccharomyces cerevisiaeKiller Toxin: Processing of the Glycosylated Precursor

1983 ◽  
Vol 3 (8) ◽  
pp. 1362-1370 ◽  
Author(s):  
H. Bussey ◽  
D. Saville ◽  
D. Greene ◽  
D. J. Tipper ◽  
K. A. Bostian
Keyword(s):  
Endoplasmic Reticulum ◽  
Secretory Pathway ◽  
Killer Toxin ◽  
Membrane System ◽  
Restrictive Temperature ◽  
Wild Type ◽  
Endoproteolytic Cleavage ◽  
Toxin Secretion

Killer toxin secretion was blocked at the restrictive temperature inSaccharomyces cerevisiae secmutants with conditional defects in theS. cerevisiaesecretory pathway leading to accumulation of endoplasmic reticulum (sec18), Golgi (sec7), or secretory vesicles (sec1). A 43,000-molecular-weight (43K) glycosylated protoxin was found by pulse-labeling in allsecmutants at the restrictive temperature. Insec18the protoxin was stable after a chase; but insec7andsec1the protoxin was unstable, and insec111K toxin was detected in cell lysates. The chymotrypsin inhibitor tosyl-l-phenylalanyl chloromethyl ketone (TPCK) blocked toxin secretion in vivo in wild-type cells by inhibiting protoxin cleavage. The unstable protoxin in wild-type and insec7andsec1cells at the restrictive temperature was stabilized by TPCK, suggesting that the protoxin cleavage was post-sec18and was mediated by a TPCK-inhibitable protease. Protoxin glycosylation was inhibited by tunicamycin, and a 36K protoxin was detected in inhibited cells. This 36K protoxin was processed, but toxin secretion was reduced 10-fold. We examined twokexmutants defective in toxin secretion; both synthesized a 43K protoxin, which was stable inkex1but unstable inkex2. Protoxin stability inkex1 kex2double mutants indicated the orderkex1→kex2in the protoxin processing pathway. TPCK did not block protoxin instability inkex2mutants. This suggested that theKEX1- andKEX2-dependent steps preceded thesec7Golgi block. We attempted to localize the protoxin inS. cerevisiaecells. Use of an in vitro rabbit reticulocyte-dog pancreas microsomal membrane system indicated that protoxin synthesized in vitro could be inserted into and glycosylated by the microsomal membranes. This membrane-associated protoxin was protected from trypsin proteolysis. Pulse-chased cells or spheroplasts, with or without TPCK, failed to secrete protoxin. The protoxin may not be secreted into the lumen of the endoplasmic reticulum, but may remain membrane associated and may require endoproteolytic cleavage for toxin secretion.

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