THE EFFECT OF OXIDATIVE STRESS INDUCED BY t-BUTYL HYDROPEROXIDE ON THE STRUCTURAL DYNAMICS OF MEMBRANE PROTEINS OF CHINESE HAMSTER FIBROBLASTS

Secretory carrier membrane proteins (SCAMPs) are ubiquitously expressed proteins of post-Golgi vesicles. In the presence of the tyrosine phosphatase inhibitor vanadate, or after overexpression in Chinese hamster ovary (CHO) cells, SCAMP1 and SCAMP3 are phosphorylated selectively on tyrosine residue(s). Phosphorylation is reversible after vanadate washout in situ or when isolated SCAMP3 is incubated with the recombinant tyrosine phosphatase PTP1B. Vanadate also causes the partial accumulation of SCAMP3, but not SCAMP1, in “patches” at or near the cell surface. A search for SCAMP kinase activities has shown that SCAMPs 1 and 3, but not SCAMP2, are tyrosine phosphorylated in EGF-stimulated murine fibroblasts overexpressing the EGF receptor (EGFR). EGF catalyzes the progressive phosphorylation of the SCAMPs up to 1 h poststimulation and may enhance colocalization of the EGFR and SCAMP3 within the cell interior. EGF also induces SCAMP–EGFR association, as detected by coimmunoprecipitation, and phosphorylation of SCAMP3 is stimulated by the EGFR in vitro. These results suggest that phosphorylation of SCAMPs, either directly or indirectly, may be functionally linked to the internalization/down-regulation of the EGFR.

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Black Rice and Rice Bran Inhibit Oxidative Stress Induced by Tert-Butyl Hydroperoxide in HepG2 Cells

Free Radical Biology and Medicine ◽

10.1016/j.freeradbiomed.2010.10.643 ◽

2010 ◽

Vol 49 ◽

pp. S221

Author(s):

Hyeonmi Ham ◽

Seon-Mi Lee ◽

Youngmin Choi ◽

Jaemin Yoon ◽

Junsoo Lee

Keyword(s):

Oxidative Stress ◽

Rice Bran ◽

Hepg2 Cells ◽

Black Rice ◽

Butyl Hydroperoxide ◽

Tert Butyl ◽

Tert Butyl Hydroperoxide

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Membrane traffic between secretory compartments is differentially affected during mitosis.

Cell Regulation ◽

10.1091/mbc.1.5.415 ◽

1990 ◽

Vol 1 (5) ◽

pp. 415-424 ◽

Cited By ~ 25

Author(s):

T Kreiner ◽

H P Moore

Keyword(s):

Plasma Membrane ◽

Membrane Proteins ◽

Secretory Pathway ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Membrane Traffic ◽

Human Growth ◽

Secretory Proteins ◽

Viral Membrane ◽

Mitotic Cells

Membrane traffic has been shown to be regulated during cell division. In particular, with the use of viral membrane proteins as markers, endoplasmic reticulum (ER)-to-Golgi transport in mitotic cells has been shown to be essentially blocked. However, the effect of mitosis on other steps in the secretory pathway is less clear, because an early block makes examination of following steps difficult. Here, we report studies on the functional characteristics of secretory pathways in mitotic mammalian tissue culture cells by the use of a variety of markers. Chinese hamster ovary cells were transfected with cDNAs encoding secretory proteins. Consistent with earlier results following viral membrane proteins, we found that the overall secretory pathway is nonfunctional in mitotic cells, and a major block to secretion is at the step between ER and Golgi: the overall rate of secretion of human growth hormone is reduced at least 10-fold in mitotic cells, and export of truncated vesicular stomatitis virus G protein from the ER is inhibited to about the same extent, as judged by acquisition of endoglycosidase H resistance. To ascertain the integrity of transport from the trans-Golgi to plasma membrane, we followed the secretion of sulfated glycosaminoglycan (GAG) chains, which are synthesized in the Golgi and thus are not subject to the earlier ER-to-Golgi block. GAG chains are valid markers for the pathway taken by constitutive secretory proteins; both protein secretion and GAG chain secretion are sensitive to treatment with n-ethyl-maleimide and monensin and are blocked at 19 degrees C. We found that the extent of GAG-chain secretion is not altered during mitosis, although the initial rate of secretion is reduced about twofold in mitotic compared with interphase cells. Thus, during mitosis, transport from the trans-Golgi to plasma membrane is much less hindered than ER-to-Golgi traffic. We conclude that transport steps are not affected to the same extent during mitosis.

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8. Evaluation of Snow Fungus Polysaccharides on benzo[a]pyrene-induced DNA Damage

Inquiry@Queen's Undergraduate Research Conference Proceedings ◽

10.24908/iqurcp.10074 ◽

2018 ◽

Author(s):

Daisy Liu

Keyword(s):

Oxidative Stress ◽

Dna Damage ◽

Free Radical ◽

Radical Scavenging ◽

Chinese Hamster ◽

Negative Control ◽

Lung Fibroblasts ◽

Protective Effects ◽

Tremella Fuciformis

Snow fungus, Tremella fuciformis, has been demonstrated to have numerous health benefits including purported chemopreventive properties due to free radical-scavenging ability. Protective effects derived from snow fungus polysaccharides are evaluated on Chinese hamster lung fibroblasts (CCL-39) exposed to carcinogen benzo[a]pyrene known to cause free radical formation and oxidative stress to cells. In this experiment, it was hypothesized that the naturally occurring polysaccharides in snow fungus are able to protect against or reduce oxidative stress-induced DNA damage. Polysaccharides were isolated through an alkaline extraction and in-vitro digestion. DNA damage was measured using the single-cell gel electrophoresis comet assay after exposure to benzo[a]pyrene and polysaccharide extract to lung fibroblasts. Results were calculated using the mean and standard deviation data of tail length and area, respectively. Each damaged cell was measured and analyzed through ImageJ Editing Software. The results indicate a promising trend which depict snow fungus polysaccharides yielding lower levels of DNA damage compared to cells exposed to benzo[a]pyrene and compared to the negative control (phosphate buffered saline and Dulbecco’s cell medium). This study suggests polysaccharides from Tremella fuciformis could truly prevent cellular DNA damage by protecting against oxidative stress.

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Potential role of mitochondrial damage and S9 mixture including metabolic enzymes in ZnO nanoparticles-induced oxidative stress and genotoxicity in Chinese hamster lung (CHL/IU) cells

Mutation Research/Genetic Toxicology and Environmental Mutagenesis ◽

10.1016/j.mrgentox.2018.07.003 ◽

2018 ◽

Vol 834 ◽

pp. 25-34 ◽

Cited By ~ 2

Author(s):

Hiroyuki Yanagisawa ◽

Yoshiko Seki ◽

Shingo Yogosawa ◽

Shota Takumi ◽

Hidesuke Shimizu ◽

...

Keyword(s):

Oxidative Stress ◽

Zno Nanoparticles ◽

Potential Role ◽

Chinese Hamster ◽

Metabolic Enzymes ◽

Mitochondrial Damage

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Protective Effects of Saponin Mixture, Isolated from <i>Astragalus monspessulanus</i> subsp. <i>monspessulanus</i> on Tert-Butyl Hydroperoxide—Induced Oxidative Stress in Isolated Rat Hepatocytes

American Journal of Plant Sciences ◽

10.4236/ajps.2015.66085 ◽

2015 ◽

Vol 06 (06) ◽

pp. 799-803 ◽

Cited By ~ 1

Author(s):

Magdalena Spasova Kondeva-Burdina ◽

Viktor Bratkov ◽

Rumyana Lubomirova Simeonova ◽

Vessela Bisserova Vitcheva ◽

Ilina Nikolaeva Krasteva ◽

...

Keyword(s):

Oxidative Stress ◽

Rat Hepatocytes ◽

Protective Effects ◽

Isolated Rat Hepatocytes ◽

Butyl Hydroperoxide ◽

Tert Butyl ◽

Tert Butyl Hydroperoxide ◽

Isolated Rat

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Monitoring mitochondrial translation by pulse SILAC

10.1101/2021.01.31.428997 ◽

2021 ◽

Author(s):

Koshi Imami ◽

Matthias Selbach ◽

Yasushi Ishihama

Keyword(s):

Oxidative Stress ◽

Amino Acids ◽

Membrane Proteins ◽

Translational Regulation ◽

Isotope Labeling ◽

Protein Complexes ◽

Complex Iii ◽

Mitochondrial Translation ◽

Mitochondrial Ribosomes ◽

Hydrophobic Nature

SummaryMitochondrial ribosomes are specialized to translate the 13 membrane proteins encoded in the mitochondrial genome, but it is challenging to quantify mitochondrial translation products due to their hydrophobic nature. Here, we introduce a proteomic method that combines biochemical isolation of mitochondria with pulse stable isotope labeling by amino acids in cell culture (pSILAC). Our method provides the highest protein coverage (quantifying 12 out of the 13 inner-membrane proteins; average 2-fold improvement over previous studies) with the shortest measurement time. We applied this method to uncover the global picture of (post)translational regulation of both mitochondrial- and nuclear-encoded proteins involved in the assembly of protein complexes that mediate oxidative phosphorylation (OXPHOS). The results allow us to infer the assembly order of complex components and/or partners, as exemplified by complex III. This method should be applicable to study mitochondrial translation programs in many contexts, including oxidative stress and mitochondrial disease.

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The Effect oftert-Butyl Hydroperoxide-Induced Oxidative Stress on Lean and Steatotic Rat HepatocytesIn Vitro

Oxidative Medicine and Cellular Longevity ◽

10.1155/2014/752506 ◽

2014 ◽

Vol 2014 ◽

pp. 1-12 ◽

Cited By ~ 50

Author(s):

Otto Kučera ◽

René Endlicher ◽

Tomáš Roušar ◽

Halka Lotková ◽

Tomáš Garnol ◽

...

Keyword(s):

Oxidative Stress ◽

High Fat Diet ◽

Respiratory Control ◽

Rat Hepatocytes ◽

Severe Damage ◽

Total Glutathione ◽

Nonalcoholic Fatty Liver ◽

Butyl Hydroperoxide ◽

Toxic Liver Injury ◽

Dependent State

Oxidative stress and mitochondrial dysfunction play an important role in the pathogenesis of nonalcoholic fatty liver disease and toxic liver injury. The present study was designed to evaluate the effect of exogenous inducer of oxidative stress (tert-butyl hydroperoxide, tBHP) on nonfatty and steatotic hepatocytes isolated from the liver of rats fed by standard and high-fat diet, respectively. In control steatotic hepatocytes, we found higher generation of ROS, increased lipoperoxidation, an altered redox state of glutathione, and decreased ADP-stimulated respiration using NADH-linked substrates, as compared to intact lean hepatocytes. Fatty hepatocytes exposed to tBHP exert more severe damage, lower reduced glutathione to total glutathione ratio, and higher formation of ROS and production of malondialdehyde and are more susceptible to tBHP-induced decrease in mitochondrial membrane potential. Respiratory control ratio of complex I was significantly reduced by tBHP in both lean and steatotic hepatocytes, but reduction in NADH-dependent state 3 respiration was more severe in fatty cells. In summary, our results collectively indicate that steatotic rat hepatocytes occur under conditions of enhanced oxidative stress and are more sensitive to the exogenous source of oxidative injury. This confirms the hypothesis of steatosis being the first hit sensitizing hepatocytes to further damage.

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