30 research outputs found

    Standardization of a simple method to study whole saliva: clinical use in different pathologies.

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    En este trabajo se describe la normatización de un método para determinar flujo salival en humanos utilizando saliva total obtenida del piso de la boca mediante un eyector dental descartable y una bomba de vacío (equipo dental). En este estudio se evaluaron 40 sujetos sanos de ambos sexos y 51 pacientes con diversas patologías (Síndrome de Sjögren, Disfunción Tiroidea, Diabetes Mellitus). Se demostró que el flujo salival basal era estable a partir de los primeros 5 minutos de colocado el eyector en la cavidad bucal. No se encontraron diferencias significativas en el flujo salival basal comparando los sexos, siendo independiente de la intensidad del vacío efectuado por la bomba. El flujo de saliva total estimulada fue determinado durante 3 minutos, luego de los primeros 5 minutos de colocado el eyector en la boca. El estímulo se efectuó adosando en la cara dorsal de la lengua discos de papel absorbente, embebidos en ácido cítrico al 2 %. El uso de este método en pacientes con Síndrome de Sjögren confirmó la reducción del flujo salival respecto a los sujetos sanos. Los pacientes hipotiroideos y con neuropatía diabética demostraron disminución del flujo salival.The present study describes a methodology to assess the salivary flow rate in humans. Whole saliva was obtained from the floor of the mouth with a plastic dental ejector and a vacuum pump. Forty healthy subjects of both sexes and 51 patients with different pathologies (Sjögren Syndrome, Thyroid Dysfunction, Diabetes Mellitus) were included in the study. It was demonstrated that basal salivary flow rate was stable five minutes after the insertion of the oral ejector. Salivary flow rate did not show significant differences between sexes and was independent of the negative pressure level of the vacuum pump. Stimulated salivary flow rate was quantified over a period of 3 minutes, starting 5 minutes after the introduction of the oral device. The stimulus was paper filter disks soaked in citric acid (2%) placed on the tongue dorsum. The use of this method confirmed the reduction of salivary flow rate in patients with Sjögren Syndrome. In addition, a significant reduction in salivary flow rate was observed in patients with primary thyroid insufficiency and peripheral neuropathy secondary to Diabetes Mellitus.Fil: Tumilasci, Omar Rene. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Fisiología; ArgentinaFil: Cardoso, Estela M. del Luján. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Contreras, Liliana Noemí. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Belforte, Juan Emilio. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Fisiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; ArgentinaFil: Arregger, Alejandro Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Ostuni, Mariano A.. Inserm; Franci

    Permeability of phospholipid membranes and human red blood cell membranes to hydrogen peroxide

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    Resumen del Conference paper presentado a SfRBM 28th Annual ConferenceHydrogen peroxide (H2O2) is an oxygen-derived oxidant involved in multiple redox processes in the cell, ranging from physiological signaling pathways to oxidative damage reactions when it is found at higher concentrations. In the vascular system, H2O2 is metabolized mainly by red blood cells (RBC) due to their very efficient antioxidant systems and high membrane permeability. However, the information regarding H2O2 transport in the human RBC membrane is limited, as neither the exact value of the permeability coefficient (Pm) nor the permeation mechanisms are known. To explore whether H2O2 permeates through the lipid fraction or protein channels, we studied H2O2 solubility in organic solvents and its permeability in lipid membranes, in order to compare with the RBC membrane. Through measurements of partition constants, we found that H2O2 is 14 and 122000 times less soluble in octanol and hexadecane than in water, anticipating a large thermodynamic barrier to H2O2 permeation by lipid membranes. The Pm in phospholipid membranes of different compositions, determined using the catalase-latency method, varied from 4×10-4 to 5×10-3 cm s-1, at 37°C. On the other hand, in human RBC we determined a Pm of 1.6×10-3 cm s-1. After obtaining these results, we evaluated the potential role of aquaporins as H2O2 transporters by checking the effect of aquaporin inhibitors in H2O2 consumption by RBC, and also by studying H2O2 permeability in RBC devoid of either aquaporin 1 or aquaporin 3. Surprisingly, we could not detect any differences in H2O2 permeability in any case. Altogether, these results provide new information on lipid membrane permeability to H2O2 and a new value for the Pm in human RBC, which was previously unknown. Additionally, they indicate that H2O2 is not transported by aquaporins in human RBC membranes, suggesting simple diffusion or a still unidentified membrane protein as a more probable pathway.ANII: ANII: FMV_1_2019_15559

    Translocator Protein-Mediated Stabilization of Mitochondrial Architecture during Inflammation Stress in Colonic Cells.

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    International audienceChronic inflammation of the gastrointestinal tract increasing the risk of cancer has been described to be linked to the high expression of the mitochondrial translocator protein (18 kDa; TSPO). Accordingly, TSPO drug ligands have been shown to regulate cytokine production and to improve tissue reconstruction. We used HT-29 human colon carcinoma cells to evaluate the role of TSPO and its drug ligands in tumor necrosis factor (TNF)-induced inflammation. TNF-induced interleukin (IL)-8 expression, coupled to reactive oxygen species (ROS) production, was followed by TSPO overexpression. TNF also destabilized mitochondrial ultrastructure, inducing cell death by apoptosis. Treatment with the TSPO drug ligand PK 11195 maintained the mitochondrial ultrastructure, reducing IL-8 and ROS production and cell death. TSPO silencing and overexpression studies demonstrated that the presence of TSPO is essential to control IL-8 and ROS production, so as to maintain mitochondrial ultrastructure and to prevent cell death. Taken together, our data indicate that inflammation results in the disruption of mitochondrial complexes containing TSPO, leading to cell death and epithelia disruption. This work implicates TSPO in the maintenance of mitochondrial membrane integrity and in the control of mitochondrial ROS production, ultimately favoring tissue regeneration

    ATPe Dynamics in Protozoan Parasites: Adapt or Perish

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    In most animals, transient increases of extracellular ATP (ATPe) are used for physiological signaling or as a danger signal in pathological conditions. ATPe dynamics are controlled by ATP release from viable cells and cell lysis, ATPe degradation and interconversion by ecto-nucleotidases, and interaction of ATPe and byproducts with cell surface purinergic receptors and purine salvage mechanisms. Infection by protozoan parasites may alter at least one of the mechanisms controlling ATPe concentration. Protozoan parasites display their own set of proteins directly altering ATPe dynamics, or control the activity of host proteins. Parasite dependent activation of ATPe conduits of the host may promote infection and systemic responses that are beneficial or detrimental to the parasite. For instance, activation of organic solute permeability at the host membrane can support the elevated metabolism of the parasite. On the other hand ecto-nucleotidases of protozoan parasites, by promoting ATPe degradation and purine/pyrimidine salvage, may be involved in parasite growth, infectivity, and virulence. In this review, we will describe the complex dynamics of ATPe regulation in the context of protozoan parasite–host interactions. Particular focus will be given to features of parasite membrane proteins strongly controlling ATPe dynamics. This includes evolutionary, genetic and cellular mechanisms, as well as structural-functional relationships.Instituto de Investigaciones Bioquímicas de La Plat

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

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    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

    Effects of Erythrocytes Treated with Alpha Hemolysin of E.Coli on Endothelial Cells

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    Uropathogenic strains of E. coli deliver the toxin alpha-hemolysin (HlyA) to optimize the host environment for the spread of infection. It was reported that at high concentrations, the toxin forms pores in eukaryotic membranes, leading to cell lysis, while lower concentrations might interfere with host-cell-signaling pathways, causing apoptosis. In the present investigation we demonstrate that a relatively low concentration of HlyA induces morphological changes and phosphatidylserine (PS) externalization of human erythrocytes. On the other hand, the unacylated nonhemolytic form of HlyA, ProHlyA induces similar morphological changes but no PS externalization. We performed osmoscan experiments to test the effect of both proteins on erythrocytes structure.Instituto de Investigaciones Bioquímicas de La PlataInstituto de Estudios Inmunológicos y Fisiopatológico

    TSPO ligands stimulate ZnPPIX transport and ROS accumulation leading to the inhibition of P. falciparum growth in human blood

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    After invading red blood cells (RBCs), Plasmodium falciparum (Pf) can export its own proteins to the host membrane and activate endogenous channels that are present in the membrane of RBCs. This transport pathway involves the Voltage Dependent Anion Channel (VDAC). Moreover, ligands of the VDAC partner TranSlocator PrOtein (TSPO) were demonstrated to inhibit the growth of the parasite. We studied the expression of TSPO and VDAC isoforms in late erythroid precursors, examined the presence of these proteins in membranes of non-infected and infected human RBCs, and evaluated the efficiency of TSPO ligands in inhibiting plasmodium growth, transporting the haem analogue Zn-protoporphyrin-IX (ZnPPIX) and enhancing the accumulation of reactive oxygen species (ROS). TSPO and VDAC isoforms are differentially expressed on erythroid cells in late differentiation states. TSPO2 and VDAC are present in the membranes of mature RBCs in a unique protein complex that changes the affinity of TSPO ligands after Pf infection. TSPO ligands dose-dependently inhibited parasite growth, and this inhibition was correlated to ZnPPIX uptake and ROS accumulation in the infected RBCs. Our results demonstrate that TSPO ligands can induce Pf death by increasing the uptake of porphyrins through a TSPO2-VDAC complex, which leads to an accumulation of ROS

    From Erythroblasts to Mature Red Blood Cells: Organelle Clearance in Mammals

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    Erythropoiesis occurs mostly in bone marrow and ends in blood stream. Mature red blood cells are generated from multipotent hematopoietic stem cells, through a complex maturation process involving several morphological changes to produce a highly functional specialized cells. In mammals, terminal steps involved expulsion of the nucleus from erythroblasts that leads to the formation of reticulocytes. In order to produce mature biconcave red blood cells, organelles and ribosomes are selectively eliminated from reticulocytes as well as the plasma membrane undergoes remodeling. The mechanisms involved in these last maturation steps are still under investigation. Enucleation involves dramatic chromatin condensation and establishment of the nuclear polarity, which is driven by a rearrangement of actin cytoskeleton and the clathrin-dependent generation of vacuoles at the nuclear-cytoplasmic junction. This process is favored by interaction between the erythroblasts and macrophages at the erythroblastic island. Mitochondria are eliminated by mitophagy. This is a macroautophagy pathway consisting in the engulfment of mitochondria into a double-membrane structure called autophagosome before degradation. Several mice knock-out models were developed to identify mitophagy-involved proteins during erythropoiesis, but whole mechanisms are not completely determined. Less is known concerning the clearance of other organelles, such as smooth and rough ER, Golgi apparatus and ribosomes. Understanding the modulators of organelles clearance in erythropoiesis may elucidate the pathogenesis of different dyserythropoietic diseases such as myelodysplastic syndrome, leukemia and anemia
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