scholarly journals Function of wild-type or mutant Rac2 and Rap1a GTPases in differentiated HL60 cell NADPH oxidase activation

Blood ◽  
1995 ◽  
Vol 85 (3) ◽  
pp. 804-811 ◽  
Author(s):  
TG Gabig ◽  
CD Crean ◽  
PL Mantel ◽  
R Rosli
Keyword(s):  
Plasma Membrane ◽  
Nadph Oxidase ◽  
Cell Line ◽  
Cell Lines ◽  
Wild Type ◽  
Free System ◽  
Hl60 Cells ◽  
Cell Free System ◽  
Nadph Oxidase Activation ◽  
O2 Production

Studies of neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation in a cell-free system showed that the low molecular-weight guanosine triphosphatase (GTPase) Rac was required, and that Rap1a may participate in activation of the catalytic complex. Full-length posttranslationally modified Rac2 was active, whereas only the 1–166 truncated form of Rap1a was functional in the cell-free system, and thus, clarification of the function of Rap1a and Rac2 in intact human phagocytes is needed to provide further insight into their roles as signal transducers from plasma membrane receptors. In the present studies, oligonucleotide-directed mutagenesis was used to introduce a series of mutations into human rap1a or rac2 in the mammalian expression vector pSR alpha neo. HL60 cells transfected with wild-type or mutated rac2 or rap1a cDNA constructs and control HL60 cells transfected with the pSR alpha neo vector containing no inserted cDNA were selected in G418-containing media, then subclones were isolated. Compared with the parent HL60 cells, each of the stable transfected cell lines differentiated similarly into neutrophil-like cells and expressed comparable levels of NADPH oxidase components p47- phox, p67-phox and gp91-phox. The differentiated vector control cell line produced O2. in response to receptor stimulation at rates that were not significantly different from parent HL60 cells. O2-. production by differentiated cell lines expressing mutated N17 Rap1a or N17 Rac2 dominant-negative proteins was inhibited, whereas O2-. production by the subline overexpressing wild-type Rap1a was increased by fourfold. O2-. production by the differentiated cell line expressing GTPase-defective V12 Rap1a was also significantly inhibited, a finding that is consistent with a requirement for cycling between guanosine diphosphate- and GTP-bound forms of Rap1a for continuous NADPH oxidase activation in intact neutrophils. A model is proposed in which Rac2 mediates assembly of the p47 and p67 oxidase components on the cytosolic face of the plasma membrane via cytoskeletal reorganization, whereas Rap1a functions downstream as the final activation switch involving direct physical interaction with the transmembrane flavocytochrome component of the NADPH oxidase.

Neutrophil p29 Peroxiredoxin VI/aiPLA2 (p29 Prdx VI) Enhances Superoxide Production by the NADPH Oxidase in a Manner Dependent On the PLA2 Active Site.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3592-3592 ◽  
Author(s):  
Michael Ellison ◽  
Gail Thurman ◽  
Daniel R. Ambruso
Keyword(s):  
Nadph Oxidase ◽  
Active Site ◽  
Respiratory Burst ◽  
The Other ◽  
Pla2 Activity ◽  
Wild Type ◽  
Free System ◽  
Oxidase System ◽  
Cell Free System ◽  
O2 Production

Abstract Abstract 3592 Poster Board III-529 Introduction An intact respiratory burst is critical to the microbial activity of neutrophils. The respiratory burst is initiated by assembly and activation of the NADPH oxidase. We have identified a 29 kDa protein from human neutrophils (p29) which binds to the NADPH oxidase component p67phox and enhances superoxide anion (O2−) production in a cell-free, NADPH activity oxidase system. The protein was identified as Prdx VI by sequence and the recombinant molecule was found to have Prdx activity. Interestingly, Prdx VI also has a calcium-independent PLA2 activity that is optimal at low pH (aiPLA2). Although Prdx VI is found in many tissues, its function in myeloid cells is not well established. p29 Prdx VI was knocked down in a myeloid cell line (PLB-985) and the effect on the respiratory burst of theses cells was investigated to determine its role in the respiratory burst. In addition, mutant or wild type p29 Prdx were introduced into the cell free system, to explore the possibility that at least one of the catalytic activities is involved in enhancing the oxidase. Methods PLB-985 cells were transfected with a plasmid encoding a p29 Prdx VI targeting shRNA or a negative control plasmid and stable transfectants were selected in puromycin containing media. Knockdown of p29 Prdx VI was confirmed by Western blot, but no change in the other known oxidase components was detected. After maturation of the knockdown and control cells by DMSO for 4 days, they were stimulated with fMLP (1μM) and O2− production was measured by luminescence of Diogenes probe. In separate experiments, neutrophil plasma membranes, recombinant p47phox, p67phox and constitutively active Rac1 were added to an assay mix. Addition of SDS assembled of the complex in this cell free system, NADPH was added and O2− production was measured as SOD inhibitable, cytochrome C reduction. O2− production when recombinant wild type p29 Prdx VI was added to the system was compared to activity caused by p29 Prdx VI mutated in the peroxiredoxin active site (C47S) or the aiPLA2 active site (D140A). Wild type and mutant p29 Prdx VI were expressed with tags in sf9 insect cells and purified by Ni2+ chelation chromatography. Results Using stable expression of shRNA, p29 Prdx VI protein was reduced to 31+/−18% (SD) of that in non-knockdown control cells but the levels of the other known oxidase components were unchanged. O2− production in response to fMLP was reduced to 51%+/−12% (SD). When added to the cell-free oxidase system, wild type p29 Prdx VI and p29 Prdx VI mutated in the peroxiredoxin active site increased the O2− production rate by similar amounts (to 323+/−94% and 417+/−125% of the assay rate in the absence of p29 Prdx VI respectively; numbers are mean ± SD for three separate experiments). Recombinant p29 mutated in the PLA2 active site however increased O2− production to only 178+/−33%. Conclusion These results indicate that p29 Prdx VI is required for optimal O2− production in intact myeloid cells. Data from the cell-free oxidase system suggest that the PLA2 activity of p29 Prdx VI, rather than its peroxiredoxin activity, account for its ability to stimulate O2- production. These studies document a specific biochemical role for p29 Prdx VI in the neutrophil respiratory burst and associated microbicidal activity. Furthermore, lipid modification by p29 Prdx VI may thus be a key step in maximal activation of the NADPH oxidase. Disclosures: No relevant conflicts of interest to declare.

Sphingosine Inhibition of NADPH Oxidase Activation in a Cell-Free System

1996 ◽  
Vol 120 (4) ◽  
pp. 705-709 ◽  
Author(s):  
J.-i. Sasaki ◽  
M. Yamaguchi ◽  
S. Saeki ◽  
H. Yamane ◽  
N. Okamura ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Free System ◽  
Cell Free System ◽  
A Cell ◽  
Nadph Oxidase Activation

scholarly journals Glucocerebroside inhibits NADPH oxidase activation in cell-free system

2004 ◽  
Vol 1688 (3) ◽  
pp. 197-203 ◽  
Author(s):  
Patryk Moskwa ◽  
Anita Palicz ◽  
Marie-Hélène Paclet ◽  
Marie-Claire Dagher ◽  
Melinda Erdős ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Free System ◽  
Cell Free System ◽  
Nadph Oxidase Activation

scholarly journals Molecular analysis of ethyl methanesulfonate-induced reversion of a chromosomally integrated mutant shuttle vector gene in mammalian cells.

1988 ◽  
Vol 8 (10) ◽  
pp. 4185-4189 ◽  
Author(s):  
J A Greenspan ◽  
F M Xu ◽  
R L Davidson
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Dna Sequences ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Shuttle Vector ◽  
Ethyl Methanesulfonate ◽  
Wild Type ◽  
Single Base ◽  
Type Sequence

The molecular mechanisms of ethyl methanesulfonate-induced reversion in mammalian cells were studied by using as a target a gpt gene that was integrated chromosomally as part of a shuttle vector. Murine cells containing mutant gpt genes with single base changes were mutagenized with ethyl methanesulfonate, and revertant colonies were isolated. Ethyl methanesulfonate failed to increase the frequency of revertants for cell lines with mutant gpt genes carrying GC----AT transitions or AT----TA transversions, whereas it increased the frequency 50-fold to greater than 800-fold for cell lines with mutant gpt genes carrying AT----GC transitions and for one cell line with a GC----CG transversion. The gpt genes of 15 independent revertants derived from the ethyl methanesulfonate-revertible cell lines were recovered and sequenced. All revertants derived from cell lines with AT----GC transitions had mutated back to the wild-type gpt sequence via GC----AT transitions at their original sites of mutation. Five of six revertants derived from the cell line carrying a gpt gene with a GC----CG transversion had mutated via GC----AT transition at the site of the original mutation or at the adjacent base in the same triplet; these changes generated non-wild-type DNA sequences that code for non-wild-type amino acids that are apparently compatible with xanthine-guanine phosphoribosyltransferase activity. The sixth revertant had mutated via CG----GC transversion back to the wild-type sequence. The results of this study define certain amino acid substitutions in the xanthine-guanine phosphoribosyltransferase polypeptide that are compatible with enzyme activity. These results also establish mutagen-induced reversion analysis as a sensitive and specific assay for mutagenesis in mammalian cells.

Growth Properties of Neural Cell Lines Immortalized with the Tsa58 Allele of Sv40 Large T Antigen

1997 ◽  
Vol 6 (3) ◽  
pp. 231-238 ◽  
Author(s):  
M.E. Truckenmiller ◽  
Ora Dillon-Carter ◽  
Carlo Tornatore ◽  
Henrietta Kulaga ◽  
Hidetoshi Takashima ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cell Line ◽  
Cell Lines ◽  
T Antigen ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Large T Antigen ◽  
Wild Type ◽  
Sv40 Large T Antigen ◽  
Growth Properties

In vitro growth properties of three CNS-derived cell lines were compared under a variety of culture conditions. The M213-20 and J30a cell lines were each derived from embryonic CNS culture with the temperature-sensitive (ts) allele of SV40 large T antigen, tsA58, while the A7 cell line was immortalized using wild-type SV40 large T antigen. Cells immortalized with tsA58 SV40 large T proliferate at the permissive temperature, 33° C, while growth is expected to be suppressed at the nonpermissive temperature, 39.5°C. Both the M213-20 and J30a cell lines were capable of proliferating at 39.5°C continuously for up to 6 mo. All three cell lines showed no appreciable differences in growth rates related to temperature over a 7-day period in either serum-containing or defined serum-free media. The percentage of cells in S-phase of the cell cycle did not decrease or was elevated at 39.5°C for all three cell lines. After 3 wk at 39.5°C, the three cell lines also showed positive immunostaining using two monoclonal antibodies reacting with different epitopes of SV40 large T antigen. Double strand DNA sequence analyses of a 300 base pair (bp) fragment of the large T gene from each cell line, which included the ts locus, revealed mutations in both the J30a and M213-20 cell lines. The J30a cell line ts mutation had reverted to wild type, and two additional loci with bp substitutions with predicted amino acid changes were also found. While the ts mutation of the M213-20 cells was retained, an additional bp substitution with a predicted amino acid change was found. The A7 cell line sequence was identical to the reference wild-type sequence. These findings suggest that (a) nucleic acid sequences in the temperature-sensitive region of the tsA58 allele of SV40 large T are not necessarily stable, and (b) temperature sensitivity of cell lines immortalized with tsA58 is not necessarily retained.

scholarly journals The mechanism of activation of NADPH oxidase in the cell-free system: the activation process is primarily catalytic and not through the formation of a stoichiometric complex

1999 ◽  
Vol 341 (2) ◽  
pp. 251-255 ◽  
Author(s):  
Andrew R. CROSS ◽  
Richard W. ERICKSON ◽  
John T. CURNUTTE
Keyword(s):  
Nadph Oxidase ◽  
Binding Site ◽  
Single Molecule ◽  
Active State ◽  
Stable Complex ◽  
Activation Process ◽  
Free System ◽  
Cell Free System ◽  
Flavocytochrome B ◽  
Stoichiometric Complex

It is commonly assumed that activation of the superoxide-generating NADPH oxidase requires the formation of a stable complex between flavocytochrome b-245 (the gp91phox/p22phox heterodimer) and the cytosolic cofactors p47phox, p67phox and Rac2. This association is thought to convert flavocytochrome b-245, which contains the NADPH-binding site, flavin and haem centres, from an inactive into an active state. Here we provide evidence that, in the cell-free system, this activation process does not necessarily require the formation of a stable stoichiometric complex between the phox proteins. To explain this data we propose the hypothesis that p67phox (and possibly Rac2), are capable of activating flavocytochrome b-245 in a catalytic fashion, where a single molecule of p67phox (or Rac2) is capable of activating multiple flavocytochrome b-245 molecules.

scholarly journals Olfactomedin 4 contributes to hydrogen peroxide-induced NADPH oxidase activation and apoptosis in mouse neutrophils

2018 ◽  
Vol 315 (4) ◽  
pp. C494-C501 ◽  
Author(s):  
Wenli Liu ◽  
Yueqin Liu ◽  
Hongzhen Li ◽  
Griffin P. Rodgers
Keyword(s):  
Hydrogen Peroxide ◽  
Nadph Oxidase ◽  
Molecular Mechanisms ◽  
Oxidase Inhibitor ◽  
Superoxide Production ◽  
Wild Type ◽  
Escherichia Coli Bacteria ◽  
Induced Apoptosis ◽  
Nadph Oxidase Activation ◽  
Deficient Mice

Neutrophils increase production of reactive oxygen species, including superoxide, hydrogen peroxide (H2O2), and hydroxyl radical, to destroy invading microorganisms under pathological conditions. Conversely, oxidative stress conditions, such as the presence of H2O2, induce neutrophil apoptosis, which helps to remove neutrophils after inflammation. However, the detailed molecular mechanisms that are involved in the latter process have not been elucidated. In this study, we investigated the potential role of olfactomedin 4 (Olfm4) in H2O2-induced superoxide production and apoptosis in mouse neutrophils. We have demonstrated that Olfm4 is not required for maximal-dosage PMA- and Escherichia coli bacteria-induced superoxide production, but Olfm4 contributes to suboptimal-dosage PMA- and H2O2-induced superoxide production. Using an NADPH oxidase inhibitor and gp91phox-deficient mouse neutrophils, we found that NAPDH oxidase was required for PMA-stimulated superoxide production and that Olfm4 mediated H2O2-induced superoxide production through NADPH oxidase, in mouse neutrophils. We have shown that neutrophils from Olfm4-deficient mice exhibited reduced H2O2-induced apoptosis compared with neutrophils from wild-type mice. We also demonstrated that neutrophils from Olfm4-deficient mice exhibited reduced H2O2-stimulated mitochondrial damage and membrane permeability, and as well as reduced caspase-3 and caspase-9 activity, compared with neutrophils from wild-type mice. Moreover, the cytoplasmic translocation of the proapoptotic mitochondrial proteins Omi/HtrA2 and Smac/DIABLO in response to H2O2was reduced in neutrophils from Olfm4-deficient mice compared with neutrophils from wild-type mice. Our study demonstrates that Olfm4 contributes to H2O2-induced NADPH oxidase activation and apoptosis in mouse neutrophils. Olfactomedin 4 might prove to be a potential target for future studies on inflammatory neutrophil biology and for inflammatory disease treatment.

Chromosome-mediated gene transfer of hydroxyurea resistance and amplification of ribonucleotide reductase activity

1983 ◽  
Vol 3 (6) ◽  
pp. 1053-1061
Author(s):  
W H Lewis ◽  
P R Srinivasan
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Ribonucleotide Reductase ◽  
Chinese Hamster ◽  
Fold Increase ◽  
Resistant Cell ◽  
Plating Efficiency ◽  
Wild Type Cell ◽  
Wild Type ◽  
Metaphase Chromosomes

Metaphase chromosomes purified from a hydroxyurea-resistant Chinese hamster cell line were able to transform recipient wild-type cells to hydroxyurea resistance at a frequency of 10(-6). Approximately 60% of the resulting transformant clones gradually lost hydroxyurea resistance when cultivated for prolonged periods in the absence of drug. One transformant was subjected to serial selection in higher concentrations of hydroxyurea. The five cell lines generated exhibited increasing relative plating efficiency in the presence of the drug and a corresponding elevation in their cellular content of ribonucleotide reductase. The most resistant cell line had a 163-fold increase in relative plating efficiency and a 120-fold increase in enzyme activity when compared with the wild-type cell line. The highly hydroxyurea-resistant cell lines had strong electron paramagnetic resonance signals characteristic of an elevated level of the free radical present in the M2 subunit of ribonucleotide reductase. Two-dimensional electrophoresis of cell-free extracts from one of the resistant cell lines indicated that a 53,000-dalton protein was present in greatly elevated quantities when compared with the wild-type cell line. These data suggest that the hydroxyurea-resistant cell lines may contain an amplification of the gene for the M2 subunit of ribonucleotide reductase.

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