scholarly journals Modifications of proteins of membrane-cytoskeleton complex and production of reactive oxygen species in erythrocytes cryopreserved with polyethylene glycol

2021 ◽  
Vol 67 (2) ◽  
pp. 44-52
Author(s):  
N.G. Zemlianskykh ◽  
◽  
L.O. Babiychuk ◽  

Protein modifications in the membrane-cytoskeleton complex (MCC) of human erythrocytes, as well as changes in the intensity of reactive oxygen species (ROS) production upon cell cryopreservation with polyethylene glycol (PEG) were investigated. The protein profile of ghosts of erythrocytes frozen with PEG has common features with both the control and cells frozen without cryoprotectant. PEG makes it possible to restrict the structural rearrangements of the main MCC proteins under the effect of extreme factors and to restrain the amount of high molecular weight polypeptide complexes induced by the protein-cross-linking reagent diamide at the control level, in contrast to cells frozen without a cryoprotectant. However, changes related to the protein peroxiredoxin 2 in ghosts of erythrocytes cryopreserved with PEG are also attributed to cells frozen without a cryoprotectant that may be associated with the activation of oxidative processes. This is evidenced by a 10-fold increase in ROS formation in erythrocytes frozen under PEG protection. Thus, upon cryopreservation of erythrocytes with PEG, certain disorders in MCC proteins may be associated with increased formation of ROS, which may contribute to the disorganization of the structural components of MCC and disrupt the stability of cryopreserved cells under physiological conditions.

2021 ◽  
Vol 56 (16) ◽  
pp. 10041-10052
Author(s):  
Laura Sánchez-Abella ◽  
Virginia Ruiz ◽  
Adrián Pérez-San Vicente ◽  
Hans-Jürgen Grande ◽  
Iraida Loinaz ◽  
...  

1993 ◽  
Vol 4 (2) ◽  
pp. 178-186 ◽  
Author(s):  
J Himmelfarb ◽  
K A Ault ◽  
D Holbrook ◽  
D A Leeber ◽  
R M Hakim

By the use of flow cytometric techniques, this prospective, randomized crossover study was designed to analyze intradialytic granulocyte reactive oxygen species (ROS) formation in whole blood with complement-activating and noncomplement-activating hollow fiber membranes. Dialysis with a complement-activating membrane resulted in a 6.5-fold increase in granulocyte hydrogen peroxide production 15 min after dialysis initiation and remained significantly elevated (P < 0.01) through the first 30 min with this membrane in comparison to both predialysis values and simultaneous values with a noncomplement-activating membrane. Further studies demonstrated that blood obtained at 15 min with a complement-activating membrane generated significantly less granulocyte ROS production in response to Staphylococcus aureus incubation than blood obtained either predialysis or at the same time in dialysis with a noncomplement-activating membrane. Both complement-activating and noncomplement-activating dialysis membranes caused slightly decreased granulocyte responsiveness to phorbol myristate acetate. It was concluded that hemodialysis with complement-activating membranes results in increased granulocyte ROS production and decreased responsiveness to S. aureus challenge during the dialysis procedure. These results document the potential role of ROS in hemodialysis-associated pathology and susceptibility to infection.


Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3589-3589 ◽  
Author(s):  
Savita Bhalla ◽  
Kevin David ◽  
Lauren Mauro ◽  
Sheila Prachand ◽  
Mint Sirisawad ◽  
...  

Abstract HDACi block cancer cell proliferation by mechanisms that involve epigenetic gene regulation leading to cell growth arrest, differentiation, and apoptosis. Bortezomib inhibits NFKB signaling and induces apoptosis. Furthermore, anti-tumor activity of HDACi and bortezomib both depend in part on reactive oxygen species (ROS)-mediated pathways. Both have activity in NHL. We reasoned that these agents may be synergistic in part due to their dependence on overlapping pathways. We investigated the biology of PCI-24781, a pan-HDACi currently in clinical trials, and bortezomib both alone, and in combination, in HL (L428) and NHL cell lines (HF1, Ramos, & SUDHL4). Cells were incubated with increasing concentrations of PCI-24781 and bortezomib (0.25–2.0μM and 2.5–20nM, respectively) for 24–72 hour (hr). Apoptosis was determined by fluorescence-activated cell sorting (FACS) using AnnexinV-FITC/propidium iodide (AnnexinV+/PI+) staining. Reactive oxygen species (ROS) were measured by oxidation of 2′7′dichlorofluorescein diacetate (H2DCFDA) to DCF and detected by FACS. Downstream targets of NFKB such as NFKB1, Myc and IL-8 were measured in Ramos using quantitative real time polymerase chain reaction (RT-PCR) following 24 hr incubation of cells with PCI-24781 and bortezomib alone, and in combination. Dose-dependent apoptosis was seen with PCI-24781 and bortezomib alone in all HL and NHL cell lines. IC70 (dose to achieve 70% AnnexinV+/PI+) was 1μM for PCI-24781 and 2μM for L428. With bortezomib, the IC50 was 10nM in Ramos, HF1, and SUDHL4 and 20 nM in L428. The combination of PCI-24781 and bortezomib resulted in synergistic apoptosis (combination index &lt;0.2) in all 3 NHL cell lines (IC80=0.25μM PCI-24781/5nM bortezomib) and L428 (IC80=0.5μM PCI-24781/10nM bortezomib) compared with minimal cell death using each agent alone at those concentrations. Furthermore, immunoblots of L428 and Ramos showed enhanced caspase 3 and caspase 8 cleavage with the combination of PCI-24781 and bortezomib compared to either agent alone, suggesting that the synergy seen was in part caspase-dependent. HL and NHL cell lines showed a 3- to 4-fold increase in ROS with PCI-24781 or bortezomib alone and in combination at 24hr. Moreover, we found that hyperacetylation of histone-3 and histone-4 on immunoblots of cells treated with combination PCI-2478/bortezomib was significantly increased compared to PCI-24781 alone. Finally, we found that in Ramos cells PC-24781/bortezomib together resulted in downregulation of NFKB targets NFKB1 and Myc, but not IL-8. We conclude that PCI-24781 and bortezomib are active in lymphoma cell lines and that the combination results in synergistic apoptosis. Apoptosis was accompanied by caspase activation and synergistic downregulation of the NFkB pathway. These data have important clinical implications for NHL and HL.


Biologia ◽  
2007 ◽  
Vol 62 (2) ◽  
Author(s):  
Aysun Ozkan

AbstractThe aim of this study was to evaluate that: (i) epirubicin-HCl (EPI) and lymphokine-activated killer (LAK) cells cytotoxicity may be mediated by free radical generation; and (ii) resistant H1299 cells may be more sensitive to combined treatment of LAK cells plus EPI than the LAK or EPI treatment alone. Viability of H1299 cells treated with EPI, LAK and LAK plus EPI was measured using the MTT test. Amount of glutathione (GSH), protein content and enzymatic activity were measured by spectrophotometer. Glutathione S-transferase (GST)-pi expression in the cells was determined by western blot analysis. LAK plus EPI combined treatment increased susceptibility of H1299 WT and H1299 EPI(R) (300-fold EPI resistant) cells to LAK cell cytotoxicity. The resistance of H1299 EPI(R) cells to EPI appears to be associated with a developed tolerance to free radicals, most likely because of a 2-fold increase in NADPH-dependent-cytochrome-P450 reductase (NADPH-CYP reductase) activity, 11-fold GST activity and 11-and 7-fold augmented selenium dependent and independent glutathione peroxidase (GSH-Px) activity, respectively. Amount of GST-pi in H1299 EPI(R) cells is statistically different from negative control and H1299 WT (p < 0.01). It is proposed that production of reactive oxygen species and hydrogen peroxide by the treatment of EPI and LAK cells can cause cytotoxicity of H1299 WT and H1299 EPI(R) cells. Superoxide dismutase, catalase, GSH-Px, GST, NADPH-CYP reductase and GSH must be considered as part of the intracellular antioxidant defense mechanism of H1299 WT and H1299 EPI(R) cells against reactive oxygen species. Combined treatment of EPI plus LAK cells caused the increasing cytotoxicity on the H1299 EPI(R) cells.


2002 ◽  
Vol 96 (4) ◽  
pp. 926-933 ◽  
Author(s):  
Yehuda Raveh ◽  
Fumito Ichinose ◽  
Pini Orbach ◽  
Kenneth D. Bloch ◽  
Warren M. Zapol

Background Sepsis is associated with an impaired pulmonary vasodilator response to inhaled nitric oxide (NO). A combination of NO and other inflammatory mediators appears to be responsible for endotoxin-induced pulmonary vascular hyporesponsiveness to inhaled NO. The authors investigated whether scavengers of reactive oxygen species could preserve inhaled NO responsiveness in endotoxin-challenged mice. Methods The vasorelaxation to inhaled NO was studied in isolated, perfused, and ventilated lungs obtained from mice 16 h after an intraperitoneal challenge with saline or 50 mg/kg Escherichia coli lipopolysaccharide. In some mice, challenge with saline or lipopolysaccharide was followed by intraperitoneal administration of N-acetylcysteine, dimethylthiourea, EUK-8, or polyethylene glycol-conjugated catalase. Results The pulmonary vasodilator response of U46619-preconstricted isolated lungs to ventilation with 0.4, 4, and 40 ppm inhaled NO in lipopolysaccharide-challenged mice was reduced to 32, 43, and 60%, respectively, of that observed in saline-challenged mice (P &lt; 0.0001). Responsiveness to inhaled NO was partially preserved in lipopolysaccharide-challenged mice treated with a single dose of N-acetylcysteine (150 or 500 mg/kg) or 20 U/g polyethylene glycol-conjugated catalase (all P &lt; 0.05 vs. lipopolysaccharide alone). Responsiveness to inhaled NO was fully preserved by treatment with either dimethylthiourea, EUK-8, two doses of N-acetylcysteine (150 mg/kg administered 3.5 h apart), or 100 U/g polyethylene glycol-conjugated catalase (all P &lt; 0.01 vs. lipopolysaccharide alone). Conclusions When administered to mice concurrently with lipopolysaccharide challenge, reactive oxygen species scavengers prevent impairment of pulmonary vasodilation to inhaled NO. Therapy with scavengers of reactive oxygen species may provide a means to preserve pulmonary vasodilation to inhaled NO in sepsis-associated acute lung injury.


1998 ◽  
Vol 141 (6) ◽  
pp. 1423-1432 ◽  
Author(s):  
Shirlee Tan ◽  
Yutaka Sagara ◽  
Yuanbin Liu ◽  
Pamela Maher ◽  
David Schubert

Reactive oxygen species (ROS) are thought to be involved in many forms of programmed cell death. The role of ROS in cell death caused by oxidative glutamate toxicity was studied in an immortalized mouse hippocampal cell line (HT22). The causal relationship between ROS production and glutathione (GSH) levels, gene expression, caspase activity, and cytosolic Ca2+ concentration was examined. An initial 5–10-fold increase in ROS after glutamate addition is temporally correlated with GSH depletion. This early increase is followed by an explosive burst of ROS production to 200–400-fold above control values. The source of this burst is the mitochondrial electron transport chain, while only 5–10% of the maximum ROS production is caused by GSH depletion. Macromolecular synthesis inhibitors as well as Ac-YVAD-cmk, an interleukin 1β–converting enzyme protease inhibitor, block the late burst of ROS production and protect HT22 cells from glutamate toxicity when added early in the death program. Inhibition of intracellular Ca2+ cycling and the influx of extracellular Ca2+ also blocks maximum ROS production and protects the cells. The conclusion is that GSH depletion is not sufficient to cause the maximal mitochondrial ROS production, and that there is an early requirement for protease activation, changes in gene expression, and a late requirement for Ca2+ mobilization.


Antioxidants ◽  
2019 ◽  
Vol 9 (1) ◽  
pp. 32 ◽  
Author(s):  
Cristian O’Flaherty ◽  
Annie Boisvert ◽  
Gurpreet Manku ◽  
Martine Culty

Peroxiredoxins (PRDXs) are antioxidant enzymes that protect cells from oxidative stress and play a role in reactive oxygen species (ROS)-mediated signaling. We reported that PRDXs are critical for human fertility by maintaining sperm viability and regulating ROS levels during capacitation. Moreover, studies on Prdx6−/− mice revealed the essential role of PRDX6 in the viability, motility, and fertility competence of spermatozoa. Although PRDXs are abundant in the testis and spermatozoa, their potential role at different phases of spermatogenesis and in perinatal germ cells is unknown. Here, we examined the expression and role of PRDXs in isolated rat neonatal gonocytes, the precursors of spermatogonia, including spermatogonial stem cells. Gene array, qPCR analyses showed that PRDX1, 2, 3, 5, and 6 transcripts are among the most abundant antioxidant genes in postnatal day (PND) 3 gonocytes, while immunofluorescence confirmed the expression of PRDX1, 2, and 6 proteins. The role of PRDXs in gonocyte viability was examined using PRDX inhibitors, revealing that the 2-Cys PRDXs and PRDX6 peroxidases activities are critical for gonocytes viability in basal condition, likely preventing an excessive accumulation of endogenous ROS in the cells. In contrast to its crucial role in spermatozoa, PRDX6 independent phospholipase A2 (iPLA2) activity was not critical in gonocytes in basal conditions. However, under conditions of H2O2-induced oxidative stress, all these enzymatic activities were critical to maintain gonocyte viability. The inhibition of PRDXs promoted a two-fold increase in lipid peroxidation and prevented gonocyte differentiation. These results suggest that ROS are produced in neonatal gonocytes, where they are maintained by PRDXs at levels that are non-toxic and permissive for cell differentiation. These findings show that PRDXs play a major role in the antioxidant machinery of gonocytes, to maintain cell viability and allow for differentiation.


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