Permeability of human red blood cell membranes to hydrogen peroxide

Resumen del Conference paper presentado a 64th Annual Meeting of the Biophysical Society, San Diego, CA. 2020Hydrogen peroxide (H2O2) and other reactive species are important physiological mediators in the vascular system. Enzymatic production of H2O2 is involved in regulating cell growth, proliferation and vasodilation. Whereas endothelial cells are important sources of H2O2, red blood cells (RBC) are considered the most important sinks of H2O2 in the vasculature. However, little is known about the permeability of their membrane to H2O2. The permeability coefficient of human RBC membranes to H2O2 was determined using the enzyme latency method, based on measuring the rate of H2O2 decomposition in lysed vs whole cells. If the passage through the membrane is the rate limiting step in H2O2 decomposition, then a difference is observed that can be used to calculate the permeability coefficient. Additional experiments were done to differentiate between simple diffusion through the lipid fraction and facilitated diffusion through protein channels. The lack of reported permeability coefficients for lipid membranes prompted us to do experiments with phospholipid-cholesterol liposome membranes that indicated that simple diffusion is a slow process. Determination of partition coefficients in different solvents mimicking different depths of the membrane indicate that the low permeability of lipid membranes to H2O2 is caused mainly by its very low solubility in the acyl region of the bilayer. The activation energy of permeation through RBC membranes suggested that protein channels were involved in facilitating H2O2 diffusion through the membrane. Inhibitors of hAQP3 and hAQP1 had no effect in H2O2 consumption rate, suggesting that other membrane proteins may be involved. Although the RBC membrane presents a significant barrier to H2O2 passage, especially in comparison with other solutes such as oxygen and nitric oxide, the permeability is still high enough to support the role of RBC as sinks of H2O2 in circulation.ANII: FMV_1_2019_15559

Detection and quantification of nitric oxide–derived oxidants in biological systems

The free radical nitric oxide (NO) exerts biological effects through the direct and reversible interaction with specific targets (e.g. soluble guanylate cyclase) or through the generation of secondary species, many of which can oxidize, nitrosate or nitrate biomolecules. The NO -derived reactive species are typically short-lived, and their preferential fates depend on kinetic and compartmentalization aspects. Their detection and quantification are technically challenging. In general, the strategies employed are based either on the detection of relatively stable end products or on the use of synthetic probes, and they are not always selective for a particular species. In this study, we describe the biologically relevant characteristics of the reactive species formed downstream from NO , and we discuss the approaches currently available for the analysis of NO , nitrogen dioxide (NO2 ), dinitrogen trioxide (N2O3), nitroxyl (HNO), and peroxynitrite (ONOO /ONOOH), as well as peroxynitrite-derived hydroxyl (HO) and carbonate anion (CO3) radicals. We also discuss the biological origins of and analytical tools for detecting nitrite (NO2), nitrate (NO3), nitrosyl–metal complexes, S-nitrosothiols, and 3-nitrotyrosine. Moreover, we highlight state– of–the–art methods, alert readers to caveats of widely used techniques, and encourage retirement of approaches that have been supplanted by more reliable and selective tools for detecting and measuring NO -derived oxidants. We emphasize that the use of appropriate analytical methods needs to be strongly grounded in a chemical and biochemical understanding of the species and mechanistic pathways involveAgencia Nacional de Investigación e Innovación FCE_1_2017_1_136043Comisión Sectorial de Investigación Científica (CSIC)Universidad de la República. Espacio Interdisciplinari

Low-temperature far-infrared ellipsometry of convergent beam

Development of an ellipsometry to the case of a coherent far infrared irradiation, low temperatures and small samples is described, including a decision of the direct and inverse problems of the convergent beam ellipsometry for an arbitrary wavelength, measurement technique and a compensating orientation of cryostat windows. Experimental results are presented: for a gold film and UBe13 single crystal at room temperature (lambda=119 um), temperature dependencies of the complex dielectric function of SrTiO3 (lambda=119, 84 and 28 um) and of YBa2Cu3O7-delta ceramic (lambda=119 um).Comment: 14 pages, 6 figure

Leukoreduction of red blood cell concentrates for transfusion

Resumen del Conference Paper presentado a SfRBM 27th Annual Conferenc

NRF2-driven miR-125B1 and miR-29B1 transcriptional regulation controls a novel anti-apoptotic miRNA regulatory network for AML survival

Transcription factor NRF2 is an important regulator of oxidative stress. It is involved in cancer progression, and has abnormal constitutive expression in acute myeloid leukaemia (AML). Posttranscriptional regulation by microRNAs (miRNAs) can affect the malignant phenotype of AML cells. In this study, we identified and characterised NRF2-regulated miRNAs in AML. An miRNA array identified miRNA expression level changes in response to NRF2 knockdown in AML cells. Further analysis of miRNAs concomitantly regulated by knockdown of the NRF2 inhibitor KEAP1 revealed the major candidate NRF2-mediated miRNAs in AML. We identified miR-125B to be upregulated and miR-29B to be downregulated by NRF2 in AML. Subsequent bioinformatic analysis identified putative NRF2 binding sites upstream of the miR-125B1 coding region and downstream of the mir-29B1 coding region. Chromatin immunoprecipitation analyses showed that NRF2 binds to these antioxidant response elements (AREs) located in the 5′ untranslated regions of miR-125B and miR-29B. Finally, primary AML samples transfected with anti-miR-125B antagomiR or miR-29B mimic showed increased cell death responsiveness either alone or co-treated with standard AML chemotherapy. In summary, we find that NRF2 regulation of miR-125B and miR-29B acts to promote leukaemic cell survival, and their manipulation enhances AML responsiveness towards cytotoxic chemotherapeutics

Fluorescent detection of hydrogen sulfide (H2S) through the formation of pyrene excimers enhances H2S quantification in biochemical systems

Hydrogen sulfide (H2S) is produced endogenously by several enzymatic pathways and modulates physiological functions in mammals. Quantification of H2S in biochemical systems remains challenging because of the presence of interferents with similar reactivity, particularly thiols. Herein, we present a new quantification method based on the formation of pyrene excimers in solution. We synthesized the probe 2-(maleimido)ethyl 4-pyrenylbutanoate (MEPB) and determined that MEPB reacted with H2S in a two-step reaction to yield the thioether-linked dimer (MEPB)2S, which formed excimers upon excitation, with a broad peak of fluorescence emission centered at 480 nm. In contrast, we found that the products formed with thiols showed peaks at 378 and 398 nm. The difference in emission between the products prevented the interference. Furthermore, we showed that the excimer fluorescence signal yielded a linear response to H2S, with a limit of detection of 54 nM in a fluorometer. Our quantification method with MEPB was successfully applied to follow the reaction of H2S with glutathione disulfide and to quantify the production of H2S from cysteine by Escherichia coli. In conclusion, this method represents an addition to the toolkit of biochemists to quantify H2S specifically and sensitively in biochemical systems.MEC: I/FVF2017/069ANII: FCE_1_2017_1_136043CSIC: I+D 2017; I+D 202

Exploiting inflammation for therapeutic gain in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy associated with <5% 5-year survival, in which standard chemotherapeutics have limited benefit. The disease is associated with significant intra- and peritumoral inflammation and failure of protective immunosurveillance. Indeed, inflammatory signals are implicated in both tumour initiation and tumour progression. The major pathways regulating PDAC-associated inflammation are now being explored. Activation of leukocytes, and upregulation of cytokine and chemokine signalling pathways, both have been shown to modulate PDAC progression. Therefore, targeting inflammatory pathways may be of benefit as part of a multi-target approach to PDAC therapy. This review explores the pathways known to modulate inflammation at different stages of tumour development, drawing conclusions on their potential as therapeutic targets in PDAC

Interleukin-17D and Nrf2 mediate initial innate immune cell recruitment and restrict MCMV infection.

Innate immune cells quickly infiltrate the site of pathogen entry and not only stave off infection but also initiate antigen presentation and promote adaptive immunity. The recruitment of innate leukocytes has been well studied in the context of extracellular bacterial and fungal infection but less during viral infections. We have recently shown that the understudied cytokine Interleukin (IL)-17D can mediate neutrophil, natural killer (NK) cell and monocyte infiltration in sterile inflammation and cancer. Herein, we show that early immune cell accumulation at the peritoneal site of infection by mouse cytomegalovirus (MCMV) is mediated by IL-17D. Mice deficient in IL-17D or the transcription factor Nuclear factor (erythroid-derived 2)-like 2 (Nrf2), an inducer of IL-17D, featured an early decreased number of innate immune cells at the point of viral entry and were more susceptible to MCMV infection. Interestingly, we were able to artificially induce innate leukocyte infiltration by applying the Nrf2 activator tert-butylhydroquinone (tBHQ), which rendered mice less susceptible to MCMV infection. Our results implicate the Nrf2/IL-17D axis as a sensor of viral infection and suggest therapeutic benefit in boosting this pathway to promote innate antiviral responses

The permeability of human red blood cell membranes to hydrogen peroxide is independent of aquaporins

Hydrogen peroxide (H2O2) not only is an oxidant but also is an important signaling molecule in vascular biology, mediating several physiological functions. Red blood cells (RBCs) have been proposed to be the primary sink of H2O2 in the vasculature because they are the main cellular component of blood with a robust antioxidant defense and a high membrane permeability. However, the exact permeability of human RBC to H2O2 is neither known nor is it known if the mechanism of permeation involves the lipid fraction or protein channels. To gain insight into the permeability process, we measured the partition constant of H2O2 between water and octanol or hexadecane using a novel double-partition method. Our results indicated that there is a large thermodynamic barrier to H2O2 permeation. The permeability coefficient of H2O2 through phospholipid membranes containing cholesterol with saturated or unsaturated acyl chains was determined to be 4 × 10−4 and 5 × 10−3 cm s−1, respectively, at 37 °C. The permeability coefficient of human RBC membranes to H2O2 at 37 °C, on the other hand, was 1.6 × 10−3 cm s−1. Different aquaporin-1 and aquaporin-3 inhibitors proved to have no effect on the permeation of H2O2. Moreover, human RBCs devoid of either aquaporin-1 or aquaporin-3 were equally permeable to H2O2 as normal human RBCs. Therefore, these results indicate that H2O2 does not diffuse into RBCs through aquaporins but rather through the lipid fraction or a still unidentified membrane protein.ANII: FCE_2017_136043ANII: FMV_2019_155597CSIC: I+D_2014_C632-348CSIC: 2018_4