Ligation of Glycophorin A Generates Reactive Oxygen Species Leading to Decreased Red Blood Cell Function.
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ABSTRACT:
SUBMITTER: Khoory J
PROVIDER: S-EPMC4718526 | biostudies-literature | 2016
REPOSITORIES: biostudies-literature
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Project description:The main function of red blood cells (RBCs) is the transport of respiratory gases along the vascular tree. To fulfill their task, RBCs are able to elastically deform in response to mechanical forces and, pass through the narrow vessels of the microcirculation. Decreased RBC deformability was observed in pathological conditions linked to increased oxidative stress or decreased nitric oxide (NO) bioavailability, like hypertension. Treatments with oxidants and with NO were shown to affect RBC deformability ex vivo, but the mechanisms underpinning these effects are unknown. In this study we investigate whether changes in intracellular redox status/oxidative stress or nitrosation reactions induced by reactive oxygen species (ROS) or NO may affect RBC deformability. In a case-control study comparing RBCs from healthy and hypertensive participants, we found that RBC deformability was decreased, and levels of ROS were increased in RBCs from hypertensive patients as compared to RBCs from aged-matched healthy controls, while NO levels in RBCs were not significantly different. To study the effects of oxidants on RBC redox state and deformability, RBCs from healthy volunteers were treated with increasing concentrations of tert-butylhydroperoxide (t-BuOOH). We found that high concentrations of t-BuOOH (? 1 mM) significantly decreased the GSH/GSSG ratio in RBCs, decreased RBC deformability and increased blood bulk viscosity. Moreover, RBCs from Nrf2 knockout (KO) mice, a strain genetically deficient in a number of antioxidant/reducing enzymes, were more susceptible to t-BuOOH-induced impairment in RBC deformability as compared to wild type (WT) mice. To study the role of NO in RBC deformability we treated RBC suspensions from human volunteers with NO donors and nitrosothiols and analyzed deformability of RBCs from mice lacking the endothelial NO synthase (eNOS). We found that NO donors induced S-nitrosation of the cytoskeletal protein spectrin, but did not affect human RBC deformability or blood bulk viscosity; moreover, under unstressed conditions RBCs from eNOS KO mice showed fully preserved RBC deformability as compared to WT mice. Pre-treatment of human RBCs with nitrosothiols rescued t-BuOOH-mediated loss of RBC deformability. Taken together, these findings suggest that NO does not affect RBC deformability per se, but preserves RBC deformability in conditions of oxidative stress.
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Project description:Mitochondrial DNA (mtDNA) is highly polymorphic, and its variations in humans may contribute to individual differences in function. Zhang and colleagues found a strikingly higher frequency of a C150T transition in the D-loop of mtDNA from centenarians and twins of an Italian population, and also demonstrated that this base substitution causes a remodeling of the mtDNA 151 replication origin in human leukocytes and fibroblasts [1]. The C150T transition is a polymorphism associated with several haplogroups. To determine whether haplogroups that carry the C150T transition display any phenotype that may be advantageous for longevity, we analyzed cybrids carrying or not the C150T transition. These cybrids were obtained by fusing cytoplasts derived from human fibroblasts with human mtDNA-less cells (ρ(0) cells). We chose for cybrid construction and analysis haplogroup-matched pairs of fibroblast strains containing or not the C150T transition. In particular, we used, as one pair of mtDNA donors, a fibroblast strain of the U3a haplogroup, carrying the C150T transition and a strain of the U-K2 haplogroup, without the C150T transition, and as another pair, fibroblasts of the J2b haplogroup, carrying the C150T transition and of the J1c haplogroup, without the C150T transition. We have found no association of respiratory capacity, mtDNA level, mitochondrial gene expression level, or growth rate with the presence of the C150T transition. However, we have found that the cybrids with haplogroups that include the C150T transition have in common a lower reactive oxygen species (ROS) production rate than the haplogroup-matched cybrids without that transition. Thus, the lower ROS production rate may be a factor in the increased longevity associated with the U and the J2 haplogroups. Of further interest, we found that cybrids with the U3a haplogroup exhibited a higher respiration rate than the other cybrids examined.
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Project description:Currently, the novel coronavirus pneumonia has been widespread globally, and there is no specific medicine. In response to the emergency, we employed bioinformatics methods to investigate the virus's pathogenic mechanism, finding possible control methods. We speculated in previous studies that E protein was associated with viral infectivity. The present study adopted the domain search techniques to analyse the E protein. According to the results, the E protein could bind iron or haem. The iron and haem bound by the E protein came from the attacked haemoglobin and phagocytes. When E protein was attached to haem, it synthesised oxygen and water into superoxide anions, hydrogen peroxide and hydroxyl radicals. When the iron-bound E protein and the haem-bound E protein worked together, they converted superoxide anions and hydrogen peroxide into oxygen and water. These were the "ROS attack" and "ROS escape" of the virus. "ROS attack" damaged the tissues or cells exposed on the surface of the virus, and "ROS escape" decomposed the superoxide anion and hydrogen peroxide that attacked the virus. When NK cells were exposed to infected cells, viruses that had not shed from the infected cells' surface damaged them through "ROS attack". In addition, lymphocytes such as T cells and B cells, which could be close to the antigen of the virus surface, were also easily damaged or killed by the "ROS attack", generating a decrease in lymphocytes. When memory B cells were exposed to the virus's surface antigen, they were also damaged by "ROS attack", resulting in the patient's re-infection. The virus applied the "ROS escape" to decompose hydrogen peroxide released by phagocytes into oxygen and water. The surrounding cells were replenished with oxygen, and the patient was in a "happy hypoxia" state. When the phagocytes swallowed the virus, the E protein converted superoxide anions into oxygen and water. In this way, the virus parasitized in the vesicles of the phagocyte. While virus was in the lysosome, the E protein generated ROS to damage nearby hydrolases. In this way, the virus parasitized the lysosome. Excessive hydroxyl free radicals destroyed the membrane structure of the lysosome, causing the hydrolase release from lysosome, autophagy of phagocytic cells and subsequent cell death. As a result, the colonizing phagocytes of the virus was associated with asymptomatic infection or retest-positive. Briefly, the virus inhibited the immune system through "ROS escape", and damaged the immune system by "ROS attack". The destruction instigated a strong cytokine storm, leading to organ failure and complications.
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Project description:Bacteria require a haem biosynthetic pathway for the assembly of a variety of protein complexes, including cytochromes, peroxidases, globins, and catalase. Haem is synthesised via a series of tetrapyrrole intermediates, including non-metallated porphyrins, such as protoporphyrin IX, which is well known to generate reactive oxygen species in the presence of light and oxygen. Staphylococcus aureus has an ancient haem biosynthetic pathway that proceeds via the formation of coproporphyrin III, a less reactive porphyrin. Here, we demonstrate, for the first time, that HemY of S. aureus is able to generate both protoporphyrin IX and coproporphyrin III, and that the terminal enzyme of this pathway, HemQ, can stimulate the generation of protoporphyrin IX (but not coproporphyrin III). Assays with hydrogen peroxide, horseradish peroxidase, superoxide dismutase, and catalase confirm that this stimulatory effect is mediated by superoxide. Structural modelling reveals that HemQ enzymes do not possess the structural attributes that are common to peroxidases that form compound I [FeIV==O]+, which taken together with the superoxide data leaves Fenton chemistry as a likely route for the superoxide-mediated stimulation of protoporphyrinogen IX oxidase activity of HemY. This generation of toxic free radicals could explain why HemQ enzymes have not been identified in organisms that synthesise haem via the classical protoporphyrin IX pathway. This work has implications for the divergent evolution of haem biosynthesis in ancestral microorganisms, and provides new structural and mechanistic insights into a recently discovered oxidative decarboxylase reaction.
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Project description:Hypertension is the most prevalent cause of cardiovascular disease and kidney failure, but only about 50% of patients achieve adequate blood pressure control, in part, due to inter-individual genetic variations in the response to antihypertensive medication. Significant strides have been made toward the understanding of the role of reactive oxygen species (ROS) in the regulation of the cardiovascular system. However, the role of ROS in human hypertension is still unclear. Polymorphisms of some genes involved in the regulation of ROS production are associated with hypertension, suggesting their potential influence on blood pressure control and response to antihypertensive medication. This review provides an update on the genes associated with the regulation of ROS production in hypertension and discusses the controversies on the use of antioxidants in the treatment of hypertension, including the antioxidant effects of antihypertensive drugs.
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Project description:Fluorescent proteins can generate reactive oxygen species (ROS) upon absorption of photons via type I and II photosensitization mechanisms. The red fluorescent proteins KillerRed and SuperNova are phototoxic proteins engineered to generate ROS and are used in a variety of biological applications. However, their relative quantum yields and rates of ROS production are unclear, which has limited the interpretation of their effects when used in biological systems. We cloned and purified KillerRed, SuperNova, and mCherry - a related red fluorescent protein not typically considered a photosensitizer - and measured the superoxide (O2•-) and singlet oxygen (1O2) quantum yields with irradiation at 561 nm. The formation of the O2•--specific product 2-hydroxyethidium (2-OHE+) was quantified via HPLC separation with fluorescence detection. Relative to a reference photosensitizer, Rose Bengal, the O2•- quantum yield (ΦO2•-) of SuperNova was determined to be 1.5 × 10-3, KillerRed was 0.97 × 10-3, and mCherry 1.2 × 10-3. At an excitation fluence of 916.5 J/cm2 and matched absorption at 561 nm, SuperNova, KillerRed and mCherry made 3.81, 2.38 and 1.65 μM O2•-/min, respectively. Using the probe Singlet Oxygen Sensor Green (SOSG), we ascertained the 1O2 quantum yield (Φ1O2) for SuperNova to be 22.0 × 10-3, KillerRed 7.6 × 10-3, and mCherry 5.7 × 10-3. These photosensitization characteristics of SuperNova, KillerRed and mCherry improve our understanding of fluorescent proteins and are pertinent for refining their use as tools to advance our knowledge of redox biology.
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Project description:Background: Blood antioxidants provide propensity to mitigate reactive oxygen species (ROS) apart from other oxidative challenges during a high-energy state of migration in night migratory songbirds. The study investigated the modulation of erythrocytes, mitochondrial abundance, hematocrit changes, and relative expression of fat transport-related genes during migration in red-headed buntings (Emberiza bruniceps). We hypothesized an increase in antioxidants along with the mitigation of mitochondria-related reactive oxygen species elevation and consequential apoptosis occurring during migration. Methods: Male red-headed buntings (n = 6) were placed under short days (8 h of light and 16 h of dark, 8L:16D)/long days (14L:10D) and photo induced to simulated non-migratory, nMig; pre-migratory, pMig; and migratory, Mig, states. Erythrocyte shape, reactive oxygen species production, mitochondrial membrane potential (MMP), reticulocyte proportion, and apoptosis were analyzed using flow cytometry and relative expression of fat metabolizing and antioxidant genes was measured by using qPCR. Results: There was a significant increase in hematocrit, erythrocyte area, and mitochondrial membrane potential. Reactive oxygen species and apoptotic erythrocyte proportion declined in the Mig state. The changes in antioxidant genes (SOD1 and NOS2), fatty acid translocase (CD36), and metabolic (FABP3, DGAT2, GOT2, and ATGL) genes showed a significant increment during the Mig state. Conclusion: These results suggested that adaptive changes occur in mitochondrial behavior and apoptosis of erythrocytes. The transition in erythrocytes, antioxidant genes, and fatty acid metabolism gene expressions suggested differences in regulatory strategies at the cellular/transcriptional level during different states of simulated migration in birds.
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Project description:Emerging evidence has suggested that pancreatic adenocarcinoma is sustained by pancreatic cancer stem cells. The present study aimed to investigate the expression patterns of the pancreatic cancer stem cell surface markers cluster of differentiation CD44 and CD24 in a pancreatic adenocarcinoma cell line, and to investigate the possible mechanisms for their radiation resistance. Flow cytometry was used to analyze the expression patterns of CD44 and CD24 in the pancreatic adenocarcinoma PANC-1 cell line. In addition, a multi-target click model was used to fit cell survival curves and determine the sensitizer enhancement ratio. The apoptosis and cycle distribution of the four cell subsets was determined using flow cytometry, and the level of reactive oxygen species (ROS) was determined using the 2',7'-dichlorofluorescin diacetate probe. The present results identified that the ratios of CD44+ and CD24+ in the sorted PANC-1 cell line were 92.0 and 4.7%, respectively. Prior to radiation, no statistically significant differences were observed among the four groups. Following treatment with 6 MV of X-rays, the rate of apoptosis was decreased in the CD44+CD24+ group compared with other subsets. The percentage of G0/G1 cells was highest in the CD44+CD24+ group compared with the three other groups, which exhibited increased radiosensitivity. In addition, the level of ROS in the CD44+CD24+ group was reduced compared with the other groups. In summary, the results of the present study indicated that CD44+CD24+ exhibited stem cell properties. The lower level of ROS and apoptosis in CD44+CD24+ cells may contribute to their resistance to radiation in pancreatic adenocarcinoma.
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