scholarly journals Modulation of Intracellular ROS and Senescence-Associated Phenotypes of Xenopus Oocytes and Eggs by Selective Antioxidants

Antioxidants ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1068
Author(s):  
Alexander A. Tokmakov ◽  
Ken-Ichi Sato
Keyword(s):  
Nadph Oxidase ◽  
Mammalian Cells ◽  
Xenopus Oocytes ◽  
Accelerated Aging ◽  
Developmental Potential ◽  
Beneficial Effects ◽  
Optimized Protocol ◽  
Intracellular Ros ◽  
Natural Organic Compound ◽  
Targeted Inhibition

Aging of oocytes and eggs diminishes their reproductive and developmental potential. It has been demonstrated previously that reactive oxygen species (ROS) contribute to accelerated aging of various cells. In the present study, we measured intracellular levels of ROS and investigated effects of several selective antioxidants (AOXs) on the viability and functional activity of aging oocytes and eggs of the African clawed frog Xenopus laevis. The fluorescent cell-permeable dye DCFDA, which is widely employed for ROS detection in cultured mammalian cells, was used to monitor ROS levels in the fresh and bench-aged oocytes and eggs by an optimized protocol. It was found that intracellular ROS contents were increased in frog oocytes and eggs aged for 48 h. It was further demonstrated using selective cell-permeable AOXs targeting different ROS-generating mechanisms, that the major source of ROS in Xenopus oocytes and eggs is the plasma membrane NADPH oxidase, and that mitochondrial generation contributes to the intracellular ROS content to a lesser extent. Targeted inhibition of NADPH oxidase with a natural organic compound apocynin reduced ROS levels significantly in Xenopus oocytes and eggs, maintained their normal phenotype and supported their functional competence. To our knowledge this is the first report concerning beneficial effects of apocynin on the isolated gamete cells, such as oocytes and eggs.

Activation of the JAK-STAT pathway by reactive oxygen species

1998 ◽  
Vol 275 (6) ◽  
pp. C1640-C1652 ◽  
Author(s):  
Amy R. Simon ◽  
Usha Rai ◽  
Barry L. Fanburg ◽  
Brent H. Cochran
Keyword(s):  
Reactive Oxygen Species ◽  
Mammalian Cells ◽  
Reactive Oxygen ◽  
Buthionine Sulfoximine ◽  
Early Response ◽  
Carcinoma Cells ◽  
Oxygen Species ◽  
Intracellular Ros ◽  
Diphenylene Iodonium ◽  
Stat Pathway

Reactive oxygen species (ROS) play an important role in the pathogenesis of many human diseases, including the acute respiratory distress syndrome, Parkinson’s disease, pulmonary fibrosis, and Alzheimer’s disease. In mammalian cells, several genes known to be induced during the immediate early response to growth factors, including the protooncogenes c- fos and c- myc, have also been shown to be induced by ROS. We show that members of the STAT family of transcription factors, including STAT1 and STAT3, are activated in fibroblasts and A-431 carcinoma cells in response to H2O2. This activation occurs within 5 min, can be inhibited by antioxidants, and does not require protein synthesis. STAT activation in these cell lines is oxidant specific and does not occur in response to superoxide- or nitric oxide-generating stimuli. Buthionine sulfoximine, which depletes intracellular glutathione, also activates the STAT pathway. Moreover, H2O2stimulates the activity of the known STAT kinases JAK2 and TYK2. Activation of STATs by platelet-derived growth factor (PDGF) is significantly inhibited by N-acetyl-l-cysteine and diphenylene iodonium, indicating that ROS production contributes to STAT activation in response to PDGF. These findings indicate that the JAK-STAT pathway responds to intracellular ROS and that PDGF uses ROS as a second messenger to regulate STAT activation.

A comparison of Hsp90α and Hsp90β interactions with cochaperones and substrates

2008 ◽  
Vol 86 (1) ◽  
pp. 37-45 ◽  
Author(s):  
Aliakbar Taherian ◽  
Patrick H. Krone ◽  
Nick Ovsenek
Keyword(s):  
Heat Shock ◽  
Mammalian Cells ◽  
Xenopus Oocytes ◽  
Stress Conditions ◽  
Differentially Expressed ◽  
Substrate Interactions ◽  
Functional Activities ◽  
Functional Differences ◽  
Hsp90 Chaperone ◽  
The Individual

Hsp90 chaperone complexes function in assembly, folding, and activation of numerous substrates. The 2 vertebrate homologues encoded by the genes hsp90a and hsp90b are differentially expressed in embryonic and adult tissues and during stress; however, it is not known whether they possess identical functional activities in chaperone complexes. This question was addressed by examining potential differences between the Hsp90 isoforms with respect to both cochaperone and substrate interactions. Epitope-tagged proteins were expressed in mammalian cells or Xenopus oocytes and subjected to immunoprecipitation with an array of cochaperones. Both isoforms were shown to participate equally in multichaperone complexes, and no significant differences in cochaperone distribution were observed. The substrates Raf-1, HSF1, Cdc37, and MEK1 interacted with both Hsp90α and Hsp90β, and the relative patterns of these interactions were not affected by heat shock. The substrate kinases c-Src, CKIIB, A-raf, and Erk interacted with both isoforms; however, significantly more Hsp90α was recovered after heat shock. The data demonstrate that Hsp90α and Hsp90β exhibit similar interactions with cochaperones, but significantly different behaviors with respect to substrate interactions under stress conditions. These results reveal both functional similarities and key functional differences in the individual members of this protein family.

scholarly journals Sequences in the human c-myc P2 promoter affect the elongation and premature termination of transcripts initiated from the upstream P1 promoter.

1992 ◽  
Vol 12 (10) ◽  
pp. 4590-4600 ◽  
Author(s):  
T Meulia ◽  
A Krumm ◽  
C Spencer ◽  
M Groudine
Keyword(s):  
Mammalian Cells ◽  
Xenopus Oocyte ◽  
Xenopus Oocytes ◽  
Premature Termination ◽  
Deletion Mutants ◽  
Potential Contribution ◽  
Germinal Vesicles ◽  
Exon 1 ◽  
P2 Promoter ◽  
Steady State Levels

A conditional block to transcription elongation provides one mechanism for controlling the steady-state levels of c-myc RNA in mammalian cells. Although prematurely terminated c-myc RNAs are not detectable in mammalian cells, truncated c-myc RNAs with 3' ends that map near the end of the first exon are transcribed from human c-myc templates injected into Xenopus oocytes germinal vesicles. A series of linker scanner and deletion mutants within the c-myc P2 promoter was tested in the Xenopus oocyte injection assay to determine the potential contribution of promoter elements to the elongation or premature termination of c-myc transcription. Although this analysis failed to identify sequences in the P2 promoter that significantly affect the elongation or termination of P2-initiated transcripts, our results suggest that sequences within the P2 promoter contribute to the premature termination of transcripts initiated at the upstream P1 promoter. A subset of these sequences is essential for the efficient elongation of P1-initiated transcripts through intrinsic sites of termination at the end of exon 1. These sequences affect P1 elongation when they are downstream of the site of initiation, and we hypothesize that they may be analogous to a class of prokaryotic elements required for antitermination.

Uptake of biotin by native Xenopus laevis oocytes

1990 ◽  
Vol 259 (3) ◽  
pp. C397-C401 ◽  
Author(s):  
H. M. Said ◽  
L. Polenzani ◽  
S. Khorchid ◽  
D. Hollander ◽  
R. Miledi
Keyword(s):  
Xenopus Laevis ◽  
Mammalian Cells ◽  
Xenopus Oocytes ◽  
Sulfhydryl Group ◽  
Maximum Velocity ◽  
Significant Inhibition ◽  
Metabolic Inhibitors ◽  
Xenopus Laevis Oocytes ◽  
Uptake System

The present study examined biotin uptake by Xenopus laevis oocytes in vitro. Uptake of low (0.03 microM) and high (10 microM) concentrations of biotin was linear with time for up to 4 h of incubation and occurred with little initial binding to oocytes. Uptake of biotin was dependent on extracellular Na+ concentration [Na+]o and was severely inhibited when Na+ was replaced by other monovalent cations [choline, tetraethylammonia, Li+, and tris(hydroxymethyl)aminomethane]. The initial rate of biotin uptake was saturable as a function of concentration with an apparent Michaelis constant of 3.9 +/- 0.5 microM and maximum velocity of 1,559 +/- 70 fmol.oocyte-1.h-1. Addition to the incubation medium of biotin structural analogues desthiobiotin and thioctic acid caused significant and concentration-dependent inhibition in the uptake of [3H]biotin. This inhibition was found to be competitive in nature with inhibition constant values of 9 and 17.5 microM. In contrast, neither the structural analogue biocytin nor biotin methyl ester (compounds in which the carboxyl group of the valeric acid moiety is blocked) showed any effect on the uptake of [3H]biotin. Biotin uptake was significantly blocked by the metabolic inhibitors dinitrophenol, cyanide, and azide and by incubation at 4 degrees C. Also, the sulfhydryl group blocker p-(chloromercuri)phenylsulfonate caused significant inhibition in biotin uptake. These results demonstrate that Xenopus oocytes possess an uptake system for biotin in its cell membrane that is Na+, energy, and temperature dependent. These characteristics of biotin uptake are similar to those reported in mammalian cells. It is suggested that Xenopus oocytes might be a useful in vitro model system to study the details of the mechanisms and regulation of biotin movement across biological membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Both the wild type and a functional isoform of CFTR are expressed in kidney

1996 ◽  
Vol 270 (6) ◽  
pp. F1038-F1048 ◽  
Author(s):  
M. M. Morales ◽  
T. P. Carroll ◽  
T. Morita ◽  
E. M. Schwiebert ◽  
O. Devuyst ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Mammalian Cells ◽  
Xenopus Oocytes ◽  
Transmembrane Domain ◽  
Renal Medulla ◽  
Regulatory Domain ◽  
Wild Type ◽  
Transmembrane Segments ◽  
Binding Domains ◽  
Cl Channels

The cystic fibrosis transmembrane conductance regulator (CFTR) consists of five domains, two transmembrane-spanning domains, each composed of six transmembrane segments, a regulatory domain, and two nucleotide-binding domains (NBDs). CFTR is expressed in kidney, but its role in overall renal function is not well understood, because mutations in CFTR found in patients with cystic fibrosis are not associated with renal dysfunction. To learn more about the distribution and functional forms of CFTR in kidney, we used a combination of molecular, cell biological, and electrophysiological approaches. These include an evaluation of CFTR mRNA and protein expression, as well as both two-electrode and patch clamping of CFTR expressed either in Xenopus oocytes or mammalian cells. In addition to wild-type CFTR mRNA, an alternate form containing only the first transmembrane domain (TMD), the first NBD, and the regulatory domain (TNR-CFTR) is expressed in kidney. Although missing the second set of TMDs and the second NBD, when expressed in Xenopus oocytes, TNR-CFTR has cAMP-dependent protein kinase A (PKA)-stimulated single Cl- channel characteristics and regulation of PKA activation of outwardly rectifying Cl- channels that are very similar to those of wild-type CFTR. TNR-CFTR mRNA is produced by an unusual mRNA processing mechanism and is expressed in a tissue-specific manner primarily in renal medulla.

Transient expression of oatp organic anion transporter in mammalian cells: identification of candidate substrates

1996 ◽  
Vol 270 (2) ◽  
pp. F319-F325 ◽  
Author(s):  
N. Kanai ◽  
R. Lu ◽  
Y. Bao ◽  
A. W. Wolkoff ◽  
V. L. Schuster
Keyword(s):  
Hela Cell ◽  
Transient Expression ◽  
Mammalian Cells ◽  
Xenopus Oocytes ◽  
Organic Anion ◽  
Expression System ◽  
Indigo Carmine ◽  
Organic Anions ◽  
Phenol Red ◽  
Cell Monolayers

The cDNA for the rat liver organic anion-transporting polypeptide "oatp" has been shown to encode transport of bromosulfophthalein (BSP) and bile salts in Xenopus oocytes (E. Jacquemin, B. Hagenbuch, B. Stieger, A. W. Wolkoff, and P. J. Meier. Proc. Natl. Acad. Sci. USA 91: 133-137, 1994). Because oatp mRNA is expressed strongly in the kidney, we sought to determine whether renal oatp might play a role in the known secretion of a large variety of organic anions by the kidney. We transiently expressed a full-length oatp cDNA, cloned in pSPORT, in HeLa cell monolayers using the recombinant vaccinia virus vtf7-3. We tested an array of organic anions as candidate substrates by determining their ability to compete with tracer BSP for transport. HeLa cell monolayers transfected with the oatp cDNA transported tracer BSP and taurocholate at rates substantially higher than monolayers transfected with a control plasmid. Thus good expression can be obtained with the vaccinia-HeLa system using a standard plasmid cloning vector. BSP transport varied as a function of the medium albumin, ionic conditions, and pH in a fashion similar to that in Xenopus oocytes. Several organic anions known to be secreted by the classic secretory pathway, including p-aminohippurate (PAH), phenol red, and indigo carmine (10 microM) failed to inhibit oatp-mediated BSP transport. Direct testing using tracers revealed no oatp-mediated transport of sulfate, urate, PAH, several eicosanoids, or unconjugated or conjugated bilirubin. On the other hand, BSP transport was inhibited by approximately 50% by 10 microM corticosterone sulfate, spironolactone, and several other steroids. We conclude that the functional properties of oatp expressed in the HeLa cell/vaccinia transient expression system are comparable to those following expression in Xenopus oocytes and that steroids are likely to represent high-affinity endogenous oatp substrates. The latter hypothesis is addressed in greater detail in a companion paper.

Aspirin-Triggered Lipoxin A4 Attenuates Lipopolysaccharide-Induced Intracellular ROS in BV2 Microglia Cells by Inhibiting the Function of NADPH Oxidase

2012 ◽  
Vol 37 (8) ◽  
pp. 1690-1696 ◽  
Author(s):  
Yan Wu ◽  
Heng Zhai ◽  
Yanping Wang ◽  
Longyan Li ◽  
Jing Wu ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Lipoxin A4 ◽  
Intracellular Ros ◽  
Bv2 Microglia

Angiopoietin-1-induced angiogenesis is modulated by endothelial NADPH oxidase

2006 ◽  
Vol 291 (4) ◽  
pp. H1563-H1572 ◽  
Author(s):  
Jian-Xiong Chen ◽  
Heng Zeng ◽  
Mayme L Lawrence ◽  
Timothy S. Blackwell ◽  
Barbara Meyrick
Keyword(s):  
Endothelial Cells ◽  
Cell Migration ◽  
Nadph Oxidase ◽  
Critical Role ◽  
Endothelial Cell Migration ◽  
Mouse Heart ◽  
Mapk Phosphorylation ◽  
Porcine Coronary Artery ◽  
Intracellular Ros ◽  
Angiopoietin 1

Reactive oxygen species (ROS) play a central role in the pathogenesis of many cardiovascular diseases, such as atherosclerosis and hypertension. Endothelial NADPH oxidase is the major source of intracellular ROS. The present study investigated the role of endothelial NADPH oxidase-derived ROS in angiopoietin-1 (Ang-1)-induced angiogenesis. Exposure of porcine coronary artery endothelial cells (PCAECs) to Ang-1 (250 ng/ml) for periods up to 30 min led to a transient and dose-dependent increase in intracellular ROS. Thirty minutes of pretreatment with the NADPH oxidase inhibitors diphenylene iodinium (DPI, 10 μM) and apocynin (200 μM) suppressed Ang-1-stimulated ROS. Pretreatment with either DPI or apocynin also significantly attenuated Ang-1-induced Akt and p44/42 MAPK phosphorylation. In addition, inhibition of NADPH oxidase significantly suppressed Ang-1-induced endothelial cell migration and sprouting from endothelial spheroids. Using mouse heart microvascular endothelial cells from wild-type (WT) mice and mice deficient in the p47phox component of NADPH oxidase (p47phox−/−), we found that although Ang-1 stimulated intracellular ROS, Akt and p42/44 MAPK phosphorylation, and cell migration in WT cells, the responses were strikingly suppressed in cells from the p47phox−/− mice. Furthermore, exposure of aortic rings from p47phox−/− mice to Ang-1 demonstrated fewer vessel sprouts than WT mice. Inhibition of the Tie-2 receptor inhibited Ang-1-induced endothelial migration and vessel sprouting. Together, our data strongly suggest that endothelial NADPH oxidase-derived ROS play a critical role in Ang-1-induced angiogenesis.

Nox2 siRNA Transfection Down Regulates VEGF Induced-Angiogenesis on Endothelial Progenitor Cells Which Are Derived From HCB

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 5188-5188
Author(s):  
Eun-Sun Yoo ◽  
Yeung-Chul Mun ◽  
Eun Mi Nam ◽  
Kyoung Eun Lee ◽  
Jung Won Huh ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Western Blot ◽  
Progenitor Cells ◽  
Endothelial Progenitor Cells ◽  
Tube Formation ◽  
Sirna Transfection ◽  
Endothelial Progenitor ◽  
Intracellular Ros ◽  
Perk Expression

Abstract Abstract 5188 Background: Reactive oxygen species (ROS) such as superoxide and H2O2 have roles signaling for molecules on angiogenesis. NADPH oxidase Nox2 (gp91phox) is a major source of ROS. Previous, we had found that Nox2-based NADPH oxidase (gp91phox)-induced ROS may play important roles on EPCs migration and proliferation by VEGF (Blood. 2009;114:Abstract 1445). In the present study, we studied the impact of down-regulation of Nox2 on intracellular ROS level, proliferation, transmigration, and in vitro tube formation of HCB derived EPCs via Nox2 siRNA transfection. Methods: Outgrowing endothelial progenitor cells were established from mononuclear cells of human cord blood (Yoo et al, Stem cells. 2003;21:228-235) using EGM-2 media in a fibronectin-coated dish. EPCs were transfected with HiPerFection transfection reagent plus Nox2 siRNA or non-targeting control siRNA and cultured for 5 hours. 100ng/ml of VEGF was added to the transfected cells and cultured for overnight. Expression of Nox2 and pERK in the Nox2 siRNA transfected EPCs were detected by western blot analysis. Intracellular ROS level was analyzed by staining with 2, 7-dichlrodihydro-fluorescein-diacetate (H2DCF-DA) and flow cytometry. Transmigration against VEGF was performed using transwell system (Costar) and in vitro tube formation was assayed using In vitro angiogenesis kit (Chemicon). Results: Intracellular ROS level was increased during endothelial progenitor cell culture which were derived from HCB by VEGF treatment. Proliferation, in vitro tube formation matrigel assay and migration assay on endothelial progenitor cells using VEGF were decreased with Nox2 siRNA transfection when compared with that of control group. In western blot data, Nox2-based NADPH oxidase (gp91phox) was increased by VEGF and decreased by Nox2 siRNA transfection. VEGF induced pERK expression was also decreased by Nox2 siRNA transfection as well. Conclusions: Based on our studies, Nox2-based NADPH oxidase (gp91phox)-induced ROS may have important roles on proliferation in HCB induced EPCs by VEGF stimulation. Furthermore, Nox2 siRNA transfection into HCB derived EPC down-regulated intracellular ROS production and pERK expression. Our data may be useful finding the new therapeutic targets for ischemic heart and ischemic limb diseases by manipulating the level of intracellular ROS via Nox2. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The Xenopus tropicalis orthologue of TRPV3 is heat sensitive

2015 ◽  
Vol 146 (5) ◽  
pp. 411-421 ◽  
Author(s):  
Beiying Liu ◽  
Feng Qin
Keyword(s):  
Mammalian Cells ◽  
Xenopus Oocytes ◽  
Transient Receptor Potential ◽  
Trp Channels ◽  
Expression System ◽  
Receptor Potential ◽  
Functional Expression ◽  
Cold Sensitivity ◽  
Mammalian Cell Lines ◽  
Transient Receptor

Thermosensitive members of the transient receptor potential (TRP) family of ion channels (thermal TRP channels) play a crucial role in mammalian temperature sensing. Orthologues of these channels are present in lower vertebrates and, remarkably, some thermal TRP orthologues from different species appear to mediate opposing responses to temperature. For example, whereas the mammalian TRPV3 channel is activated by heat, frog TRPV3 is reportedly activated by cold. Intrigued by the potential implications of these opposing responses to temperature for the mechanism of temperature-dependent gating, we cloned Xenopus laevis TRPV3 and functionally expressed it in both mammalian cell lines and Xenopus oocytes. We found that, when expressed in mammalian cells, the recombinant channel lacks the reported cold sensitivity; rather, it is activated by temperatures >50°C. Furthermore, when expressed in mammalian cells, the frog orthologue shows other features characteristic of mammalian TRPV3, including activation by the agonist 2-aminoethoxydiphenyl borate and an increased response with repeated stimulation. We detected both heat- and cold-activated currents in Xenopus oocytes expressing the recombinant frog TRPV3 channel. However, cold-activated currents were also apparent in control oocytes lacking recombinant TRPV3. Our data indicate that frog TRPV3 resembles its mammalian orthologues in terms of its thermosensitivity and is intrinsically activated by heat. Thus, all known vanilloid receptors are activated by heat. Our data also show that Xenopus oocytes contain endogenous receptors that are activated by cold, and suggest that cold sensitivity of TRP channels established using Xenopus oocytes as a functional expression system may need to be revisited.

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