RESUMEN
Understanding the mechanisms by which drugs interact with cell membranes is crucial for unraveling the underlying biochemical and biophysical processes that occur on the surface of these membranes. Our research focused on studying the interaction between an ester-type derivative of tristearoyl uridine and model cell membranes composed of lipid monolayers at the air-water interface. For that, we selected a specific lipid to simulate nontumorigenic cell membranes, namely 1,2-dihexadecanoyl-sn-glycero-3-phospho-l-serine. We noted significant changes in the surface pressure-area isotherms, with a noticeable shift towards larger areas, which was lower than expected for ideal mixtures, indicating monolayer condensation. Furthermore, the viscoelastic properties of the interfacial film demonstrated an increase in both the elastic and viscous parameters for the mixed film. We also observed structural alterations using vibrational spectroscopy, which revealed an increase in the all-trans to gauche conformers ratio. This confirmed the stiffening effect of the prodrug on the lipid monolayer. In summary, this study indicates that this lipophilic prodrug significantly impacts the lipid monolayer's thermodynamic, rheological, electrical, and molecular characteristics. This information is crucial for understanding how the drug interacts with specific sites on the cellular membrane. It also has implications for drug delivery, as the drug's passage into the cytosol may involve traversing the lipid bilayer.
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Membrana Celular , Profármacos , Uridina , Profármacos/química , Profármacos/farmacología , Profármacos/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Uridina/química , Uridina/farmacología , Fosfatidilserinas/química , Termodinámica , Propiedades de Superficie , Viscosidad , ElasticidadRESUMEN
Matrix vesicles are a special class of extracellular vesicles thought to actively contribute to both physiologic and pathologic mineralization. Proteomic studies have shown that matrix vesicles possess high amounts of annexin A5, suggesting that the protein might have multiple roles at the sites of calcification. Currently, Annexin A5 is thought to promote the nucleation of apatitic minerals close to the inner leaflet of the matrix vesicles' membrane enriched in phosphatidylserine and Ca2+. Herein, we aimed at unravelling a possible additional role of annexin A5 by investigating the ability of annexin A5 to adsorb on matrix-vesicle biomimetic liposomes and Langmuir monolayers made of dipalmitoylphosphatidylserine (DPPS) and dipalmitoylphosphatidylcholine (DPPC) in the absence and in the presence of Ca2+. Differential scanning calorimetry and dynamic light scattering measurements showed that Ca2+ at concentrations in the 0.5-2.0 mM range induced the aggregation of liposomes probably due to the formation of DPPS-enriched domains. However, annexin A5 avoided the aggregation of liposomes at Ca2+ concentrations lower than 1.0 mM. Surface pressure versus surface area isotherms showed that the adsorption of annexin A5 on the monolayers made of a mixture of DPPC and DPPS led to a reduction in the area of excess compared to the theoretical values, which confirmed that the protein favored attractive interactions among the membrane lipids. The stabilization of the lipid membranes by annexin A5 was also validated by recording the changes with time of the surface pressure. Finally, fluorescence microscopy images of lipid monolayers revealed the formation of spherical lipid-condensed domains that became unshaped and larger in the presence of annexin A5. Our data support the model that annexin A5 in matrix vesicles is recruited at the membrane sites enriched in phosphatidylserine and Ca2+ not only to contribute to the intraluminal mineral formation but also to stabilize the vesicles' membrane and prevent its premature rupture.
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Anexinas , Liposomas , Anexina A5/química , Anexina A5/metabolismo , Fosfatidilserinas/química , Fosfatidilserinas/metabolismo , Biomimética , Proteómica , Calcio/metabolismoRESUMEN
The aim of this study was to evaluate the effect of supplementing bovine semen freezing extender with different concentrations of iodixanol on post-thaw sperm characteristics. Six ejaculates of three Nellore bulls were pooled and diluted in commercial extender (BotuBov®) and then divided into 4 groups: control group (without adding iodixanol); groups G1.5, G3, or G6 according to the concentration of iodixanol solution (RedCushion®). After dilution, the samples were cooled and frozen. Post-thaw semen evaluation included sperm motility by CASA immediately after thawing and after 60 min of incubation at 37°C, flow cytometry analysis for integrity of plasma and acrosomal membranes, membrane destabilization and translocation of phosphatidylserine, mitochondrial membrane potential, and formation of intracellular anion superoxide ( O 2 - ), hydrogen peroxide (H2 O2 ), and membrane lipid peroxidation. The group G6 presented significantly higher (p < .05) total and progressive motility, percentage of plasma and acrosomal membrane integrity, and H2 O2 than control and group G1.5. Furthermore, group G6 showed lower (p < .05) lipid peroxidation than control. In addition, regardless of the concentration used, the percentage of spermatozoa without phosphatidylserine translocation was higher (p < .05) in all iodixanol supplemented groups. In conclusion, iodixanol supplementation preserved the motility and integrity of sperm membranes during cryopreservation and protected against lipid peroxidation.
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Preservación de Semen , Semen , Masculino , Animales , Bovinos , Congelación , Antioxidantes/farmacología , Fosfatidilserinas , Motilidad Espermática , Preservación de Semen/veterinaria , Crioprotectores/farmacología , Espermatozoides , Análisis de Semen/veterinaria , Criopreservación/veterinaria , Suplementos DietéticosRESUMEN
BACKGROUND: The tick Amblyomma sculptum is the major vector of Rickettsia rickettsii, the causative agent of the highly lethal Brazilian spotted fever. It has been shown that R. rickettsii inhibits apoptosis in both human endothelial cells and tick cells. Apoptosis is regulated by different factors, among which inhibitors of apoptosis proteins (IAPs) play a central role. In the study reported here, we selected an IAP of A. sculptum that has not yet been characterized to assess its role in cell death and to determine the effects of its gene silencing on tick fitness and R. rickettsii infection. METHODS: An A. sculptum cell line (IBU/ASE-16) was treated with specific double-stranded RNA (dsRNA) for either IAP (dsIAP) or green fluorescent protein (dsGFP; as a control). The activity of caspase-3 and the exposure of phosphatidylserine were determined in both groups. In addition, unfed adult ticks, infected or not infected with R. rickettsii, were treated with either dsIAP or dsGFP and allowed to feed on noninfected rabbits. In parallel, noninfected ticks were allowed to feed on an R. rickettsii-infected rabbit. Ticks (infected or not with R. rickettsii) that remained unfed were used as a control. RESULTS: Caspase-3 activity and the externalization of phosphatidylserine were significantly higher in IBU/ASE-16 cells treated with dsIAP than in those treated with dsGFP. The mortality rates of ticks in the dsIAP group were much higher than those in the dsGFP group when they were allowed to feed on rabbits, independent of the presence of R. rickettsii. Conversely, lower mortality rates were recorded in unfed ticks. CONCLUSIONS: Our results show that IAP negatively regulates apoptosis in A. sculptum cells. Moreover, IAP-silenced ticks experienced higher mortality rates following the acquisition of a blood meal, suggesting that feeding may trigger the activation of apoptosis in the absence of this physiological regulator. These findings indicate that IAP is a potential antigen for an anti-tick vaccine.
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Ixodidae , Fiebre Maculosa de las Montañas Rocosas , Garrapatas , Animales , Humanos , Conejos , Garrapatas/microbiología , Amblyomma , Caspasa 3/metabolismo , Ixodidae/genética , Proteínas Inhibidoras de la Apoptosis/metabolismo , Células Endoteliales , Fosfatidilserinas/metabolismo , Rickettsia rickettsii/fisiología , BrasilRESUMEN
Clostridioides difficile (C. difficile) produces toxins A (TcdA) and B (TcdB), both associated with intestinal damage and diarrhea. Pannexin-1 (Panx1) channels allows the passage of messenger molecules, such as adenosine triphosphate (ATP), which in turn activate the P2X7 receptors (P2X7R) that regulate inflammation and cell death in inflammatory bowel diseases. The aim of this study was to verify the effect of C. difficile infection (CDI) in the expression of Panx1 and P2X7R in intestinal tissues of mice, as well as their role in cell death and IL-6 expression induced by TcdA and TcdB in enteric glial cells (EGCs). Male C57BL/6 mice (8 weeks of age) were infected with C. difficile VPI10463, and the control group received only vehicle per gavage. After three days post-infection (p.i.), cecum and colon samples were collected to evaluate the expression of Panx1 by immunohistochemistry. In vitro, EGCs (PK060399egfr) were challenged with TcdA or TcdB, in the presence or absence of the Panx1 inhibitor (10Panx trifluoroacetate) or P2X7R antagonist (A438079), and Panx1 and P2X7R expression, caspase-3/7 activity and phosphatidylserine binding to annexin-V, as well as IL-6 expression were assessed. CDI increased the levels of Panx1 in cecum and colon of mice compared to the control group. Panx1 inhibitor decreased caspase-3/7 activity and phosphatidylserine-annexin-V binding, but not IL-6 gene expression in TcdA and TcdB-challenged EGCs. P2X7 receptor antagonist accentually reduced caspase-3/7 activity, phosphatidylserine-annexin-V binding, and IL-6 gene expression in TcdA and TcdB-challenged EGCs. In conclusion, Panx1 is increased during CDI and plays an important role in the effects of C. difficile toxins in EGCs, participating in cell death induced by both toxins by promoting caspase-3/7 activation via P2X7R, which is also involved in IL-6 expression induced by both toxins.
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Toxinas Bacterianas , Clostridioides difficile , Conexinas , Proteínas del Tejido Nervioso , Receptores Purinérgicos P2X7 , Animales , Anexinas , Toxinas Bacterianas/metabolismo , Caspasa 3/metabolismo , Muerte Celular , Conexinas/genética , Conexinas/metabolismo , Mediadores de Inflamación , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuroglía/metabolismo , Fosfatidilserinas , Receptores Purinérgicos P2X7/genéticaRESUMEN
Angiotensin II (Ang II) regulates blood volume and stimulates erythropoiesis through AT1 (ATR1) and AT2 (ATR2) receptors, found in multiple tissues, including erythrocytes. Sickle cell disease (SCD) patients present altered Ang II levels. Hemoglobin S polymerization, deformability and phosphatidylserine translocation are important features of mature erythrocytes, therefore, our hypothesis is Ang II affects these parameters and, if it does, what would be the influence of AT1R and AT2R on these effects. A polymerization assay (PA), deformability, and annexin V binding were performed in SCD erythrocytes samples adding Ang II, ATR1 antagonist (losartan or eprosartan), and ATR2 antagonist (PD123319). Through the PA test, we observed a dose-dependent polymerization inhibition effect when comparing Ang II to control. Losartan did not affect the level or the rate of Ang II inhibition, while PD123319 showed an increased level of protection against polymerization, and eprosartan brought levels back to control. Ang II was able to reduce the translocation of phosphatidylserine from the inner to the outer leaflet, a marker of eryptosis, in the presence of PD123319. Also, ATR1 showed a positive effect increasing deformability. Our data shows that ATR1 is important for maintenance of erythrocyte physiological function in SCD and for prolonging its life.
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Anemia de Células Falciformes , Losartán , Acrilatos , Angiotensina II/metabolismo , Angiotensina II/farmacología , Anexina A5 , Eritrocitos/metabolismo , Hemoglobina Falciforme/metabolismo , Humanos , Imidazoles , Losartán/farmacología , Fosfatidilserinas , Polimerizacion , Receptor de Angiotensina Tipo 1/metabolismo , TiofenosRESUMEN
The main form of control of leishmaniasis is the treatment, however various side effects and poor efficacy are associated with presently available drugs. The investigation of bioactive natural products for new antileishmanial drugs is a valid approach. The present study reports the in vitro efficacy of natural isoflavonoids and terpenes against Leishmania infantum and L. amazonensis and their cytotoxicity against HepG2 cells. L. infantum and L. amazonensis promastigotes were exposed to the terpenes kaurenoic acid, xylopic acid, and (-)-α-bisabolol and to the isoflavonoids (-)-duartin and (3R)-claussequinone for antileishmanial activity and to cytotoxicity to HepG2 cells. The most effective substance against both L. infantum and L. amazonensis species was (3R)-claussequinone (IC50 = 3.21 µg/mL and 2.47 µg/mL, respectively) that disclosed low cytotoxicity against HepG2 cells (CC50 = 387.79 µg/mL). The efficacy of (3R)-claussequinone against intracellular amastigotes of L. infantum and the externalization of phosphatidylserine in promastigotes of this isoflavanoid were investigated by infection of Raw 264.7 macrophages and marking with Annexin V-FITC and propidium Iodide for flow cytometry analysis. The results for amastigotes showed that (3R)-claussequinone was able to reduce the rate of infection with IC50 = 4.61 µg/mL and did not alter the externalization of phosphatidylserine. In conclusion it is presently reported, for the first time, the striking antileishmanial activity of (3R)-claussequinone against L. infantum and L. amazonensis associated to low cytotoxicity. Furthermore, these results suggest that (3R)-claussequinone is a new hit aiming to develop new therapeutic alternatives.
Asunto(s)
Antiprotozoarios , Productos Biológicos , Leishmania infantum , Ratones , Animales , Terpenos/farmacología , Fosfatidilserinas , Propidio , Ratones Endogámicos BALB C , Antiprotozoarios/toxicidad , Antiprotozoarios/uso terapéutico , Productos Biológicos/farmacologíaRESUMEN
The biochemical machinery involved in matrix vesicles-mediated bone mineralization involves a specific set of lipids, enzymes, and proteins. Annexins, among their many functions, have been described as responsible for the formation and stabilization of the matrix vesicles' nucleational core. However, the specific role of each member of the annexin family, especially in the presence of type-I collagen, remains to be clarified. To address this issue, in vitro mineralization was carried out using AnxA6 (in solution or associated to the proteoliposomes) in the presence or in the absence of type-I collagen, incubated with either amorphous calcium phosphate (ACP) or a phosphatidylserine-calcium phosphate complex (PS-CPLX) as nucleators. Proteoliposomes were composed of 1,2-dipalmitoylphosphatidylcholine (DPPC), 1,2-dipalmitoylphosphatidylcholine: 1,2-dipalmitoylphosphatidylserine (DPPC:DPPS), and DPPC:Cholesterol:DPPS to mimic the outer and the inner leaflet of the matrix vesicles membrane as well as to investigate the effect of the membrane fluidity. Kinetic parameters of mineralization were calculated from time-dependent turbidity curves of free Annexin A6 (AnxA6) and AnxA6-containing proteoliposomes dispersed in synthetic cartilage lymph. The chemical composition of the minerals formed was investigated by Fourier transform infrared spectroscopy (FTIR). Free AnxA6 and AnxA6-proteoliposomes in the presence of ACP were not able to propagate mineralization; however, poorly crystalline calcium phosphates were formed in the presence of PS-CPLX, supporting the role of annexin-calcium-phosphatidylserine complex in the formation and stabilization of the matrix vesicles' nucleational core. We found that AnxA6 lacks nucleation propagation capacity when incorporated into liposomes in the presence of PS-CPLX and type-I collagen. This suggests that AnxA6 may interact either with phospholipids, forming a nucleational core, or with type-I collagen, albeit less efficiently, to induce the nucleation process.
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Anexina A6 , Calcinosis , 1,2-Dipalmitoilfosfatidilcolina/química , Anexina A6/metabolismo , Colágeno/metabolismo , Humanos , Fosfatos/metabolismo , Fosfatidilserinas/química , ProteolípidosRESUMEN
OBJECTIVES: The American Society of Anesthesiologists Physical Status (ASA-PS) is a grading system routinely adopted worldwide by physicians to classify patients' overall health status. Concerns have been raised surrounding the subjectiveness of this system, potentially leading to poor inter-rater agreement/reliability. We hypothesized that physicians are overconfident when assigning ASA-PS scores and that presenting them with the ASA-PS definitions/examples would improve accuracy. We therefore evaluated participants' accuracy and self-reported confidence on the ASA-PS Classification System (1) while assigning ASA-PS according to their baseline knowledge/judgment; and (2) after a single exposure to the ASA-PS definitions/examples. DESIGN: Prospective before-and-after web-based study. PARTICIPANTS: 272 anesthesiologists and 114 non-anesthesiologists. INTERVENTIONS: Participants voluntarily answered a web-based questionnaire consisting of 10 hypothetical cases. They were asked to assign an ASA-PS score and rate their perceived self-confidence level (20-100%) on the accuracy of their assigned score for each case both (1) before and (2) after reviewing the ASA-PS definitions/examples. The correct ASA-PS for each hypothetical case was determined by consensus among investigators. MEASUREMENTS: Participants' accuracy, self-reported confidence, and calibration of confidence on the application of ASA-PS Classification System. Agreement between measures was tested using kappa coefficient. RESULTS: Anesthesiologists had better accuracy than non-anesthesiologists both on initial [6(5-7) vs. 4(3-5) out of 10; p < 0.001] as well as subsequent [7(6-8) vs. 6(4-7); p < 0.001] ASA-PS score assignments. Participants' self-reported confidence was greater than their accuracy for assigned ASA-PS scores (p < 0.001). ASA-PS agreement between anesthesiologists and non-anesthesiologists was poor (κ < 0.20). Participants' accuracy for hypothetical cases of ASA-PS I, II, and III involving adult patients was overall greater than for ASA-PS IV, V, and III (the latter involving a neonate) for both anesthesiologists and non-anesthesiologists (p < 0.001). CONCLUSIONS: Physicians tend to disagree and be overconfident when assigning ASA-PS scores. A brief consultation of the ASA-PS definitions/examples improves the accuracy for both anesthesiologists and non-anesthesiologists.
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Anestesiólogos , Adulto , Azidas , Humanos , Recién Nacido , Fosfatidilserinas , Estudios Prospectivos , Reproducibilidad de los Resultados , AutoinformeRESUMEN
Leishmaniasis is a group of neglected diseases caused by parasites of the Leishmania genus. The treatment of Leishmaniasis represents a great challenge, because the available drugs present high toxicity and none of them is fully effective. Caryocar is a botanical genus rich in phenolic compounds, which leaves extracts have already been described by its antileishmanial action. Thus, we investigated the effect of pulp and peel extracts of the Caryocar coriaceum fruit on promastigote and amastigote forms of Leishmania amazonensis. Both extracts had antipromastigote effect after 24, 48, and 72 h, and this effect was by apoptosis-like process induction, with reactive oxygen species (ROS) production, damage to the mitochondria and plasma membrane, and phosphatidylserine exposure. Knowing that the fruit extracts did not alter the viability of macrophages, we observed that the treatment reduced the infection of these cells. Thereafter, in the in vitro infection context, the extracts showed antioxidant proprieties, by reducing NO, ROS, and MDA levels. Besides, both peel and pulp extracts up-regulated Nrf2/HO-1/Ferritin expression and increase the total iron-bound in infected macrophages, which culminates in a depletion of available iron for L. amazonensis replication. In silico, the molecular modeling experiments showed that the three flavonoids presented in the C. coriaceum extracts can act as synergistic inhibitors of Leishmania proteins, and compete for the active site. Also, there is a preference for rutin at the active site due to its greater interaction binding strength.Communicated by Ramaswamy H. Sarma.
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Antiprotozoarios , Leishmania , Leishmaniasis , Malpighiales , Animales , Antioxidantes/farmacología , Antiprotozoarios/farmacología , Ferritinas/metabolismo , Ferritinas/farmacología , Ferritinas/uso terapéutico , Flavonoides/farmacología , Frutas , Humanos , Hierro/metabolismo , Leishmaniasis/tratamiento farmacológico , Malpighiales/metabolismo , Ratones , Ratones Endogámicos BALB C , Factor 2 Relacionado con NF-E2/metabolismo , Fosfatidilserinas/metabolismo , Fosfatidilserinas/farmacología , Fosfatidilserinas/uso terapéutico , Especies Reactivas de Oxígeno/metabolismo , Rutina/farmacología , Rutina/uso terapéuticoRESUMEN
INTRODUCTION: Anti-phospatidylserine/prothrombin (aPS/PT) antibodies have been described in cutaneous Polyarteritis Nodosa (PAN) in association with specific manifestations. OBJECTIVES: To determine aPS/PT antibodies in patients with PAN and its correlation with clinical manifestations. METHODS: Cross-sectional comparative study including PAN patients and 20 controls (10 Microscopic Polyangiitis [MPA] and 10 Behçet's disease [BD]). Clinical and demographic variables, treatment, serologic markers, prognosis, activity and damage indexes were evaluated. aPS/PT, anti-cardiolipin (aCL), anti-beta 2 glycoprotein 1 (anti-B2GP1) antibodies, and lupus anticoagulant (LA) were determined. RESULTS: Fourteen patients with PAN were included, 11 (79%) women, with disease duration of 207 months, and mostly inactive disease. Only one patient with PAN and one with BD were positive for aPS/PT IgG. LA was the most frequent antibody identified. One patient with MPA and one with BD were positive for aCL IgM; one with MPA for anti-B2GP1 IgG, and one with PAN for anti-B2GP1 IgM. CONCLUSIONS: aPS/PT antibodies are not frequent in patients with longstanding inactive PAN.
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Poliarteritis Nudosa , Protrombina , Anticuerpos Antifosfolípidos , Estudios Transversales , Femenino , Humanos , FosfatidilserinasRESUMEN
Quinoline and 1,2,3-triazoles are well-known nitrogen-based heterocycles presenting diverse pharmacological properties, although their antileishmanial activity is still poorly exploited. As an effort to contribute with studies involving these interesting chemical groups, in the present study, a series of compounds derived from 4-aminoquinoline and 1,2,3-triazole were synthetized and biological studies using L. amazonensis species were performed. The results pointed that the derivative 4, a hybrid of 4-aminoquinoline/1,2,3-triazole exhibited the best antileishmanial action, with inhibitory concentration (IC50) values of ~1 µM against intramacrophage amastigotes of L. amazonensis , and being 16-fold more active to parasites than to the host cell. The mechanism of action of derivative 4 suggest a multi-target action on Leishmania parasites, since the treatment of L. amazonensis promastigotes caused mitochondrial membrane depolarization, accumulation of ROS products, plasma membrane permeabilization, increase in neutral lipids, exposure of phosphatidylserine to the cell surface, changes in the cell cycle and DNA fragmentation. The results suggest that the antileishmanial effect of this compound is primarily altering critical biochemical processes for the correct functioning of organelles and macromolecules of parasites, with consequent cell death by processes related to apoptosis-like and necrosis. No up-regulation of reactive oxygen and nitrogen intermediates was promoted by derivative 4 on L. amazonensis -infected macrophages, suggesting a mechanism of action independent from the activation of the host cell. In conclusion, data suggest that derivative 4 presents selective antileishmanial effect, which is associated with multi-target action, and can be considered for future studies for the treatment against disease.
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Aminoquinolinas/farmacología , Antiprotozoarios/farmacología , Leishmania mexicana/efectos de los fármacos , Triazoles/farmacología , Aminoquinolinas/síntesis química , Animales , Antiprotozoarios/química , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Membrana Celular/química , Membrana Celular/metabolismo , Fragmentación del ADN/efectos de los fármacos , Femenino , Metabolismo de los Lípidos/efectos de los fármacos , Macrófagos/parasitología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Orgánulos/efectos de los fármacos , Fosfatidilserinas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Triazoles/síntesis químicaRESUMEN
Programmed cell death (PCD) is an essential process for the immune system's development and homeostasis, enabling the remotion of infected or unnecessary cells. There are several PCD's types, depending on the molecular mechanisms, such as non-inflammatory or pro-inflammatory. Hemocytes are the main component of cellular immunity in bivalve mollusks. Numerous infectious microorganisms produce toxins that impair hemocytes functions, but there is little knowledge on the role of PCD in these cells. This study aims to evaluate in vitro whether marine toxins induce a particular type of PCD in hemocytes of the bivalve mollusk Crassostrea gigas during 4 h at 25°C. Hemocytes were incubated with two types of marine toxins: non-proteinaceous toxins from microalgae (saxitoxin, STX; gonyautoxins 2 and 3, GTX2/3; okadaic acid/dynophysistoxin-1, OA/DTX-1; brevetoxins 2 and 3, PbTx-2,-3; brevetoxin 2, PbTx-2), and proteinaceous extracts from bacteria (Vibrio parahaemolyticus, Vp; V. campbellii, Vc). Also, we used the apoptosis inducers, staurosporine (STP), and camptothecin (CPT). STP, CPT, STX, and GTX 2/3, provoked high hemocyte mortality characterized by apoptosis hallmarks such as phosphatidylserine translocation into the outer leaflet of the cell membrane, exacerbated chromatin condensation, DNA oligonucleosomal fragments, and variation in gene expression levels of apoptotic caspases 2, 3, 7, and 8. The mixture of PbTx-2,-3 also showed many apoptosis features; however, they did not show apoptotic DNA oligonucleosomal fragments. Likewise, PbTx-2, OA/DTX-1, and proteinaceous extracts from bacteria Vp, and Vc, induced a minor degree of cell death with high gene expression of the pro-inflammatory initiator caspase-1, which could indicate a process of pyroptosis-like PCD. Hemocytes could carry out both PCD types simultaneously. Therefore, marine toxins trigger PCD's signaling pathways in C. gigas hemocytes, depending on the toxin's nature, which appears to be highly conserved both structurally and functionally.
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Apoptosis/efectos de los fármacos , Toxinas Bacterianas/toxicidad , Crassostrea/efectos de los fármacos , Hemocitos/efectos de los fármacos , Toxinas Marinas/toxicidad , Animales , Toxinas Bacterianas/aislamiento & purificación , Caspasas/metabolismo , Ensamble y Desensamble de Cromatina/efectos de los fármacos , Crassostrea/inmunología , Crassostrea/metabolismo , Roturas del ADN de Doble Cadena , Hemocitos/inmunología , Hemocitos/metabolismo , Hemocitos/patología , Fosfatidilserinas/metabolismo , Vibrio/metabolismo , Vibrio parahaemolyticus/metabolismoRESUMEN
BACKGROUND: Leishmaniasis is a neglected tropical disease caused by protozoan parasites of the Leishmania genus. Currently, the treatment has limited effectiveness and high toxicity, is expensive, requires long-term treatment, induces significant side effects, and promotes drug resistance. Thus, new therapeutic strategies must be developed to find alternative compounds with high efficiency and low cost. Solidagenone (SOL), one of the main constituents of Solidago chilensis, has shown gastroprotective, anti-inflammatory and immunomodulatory effects. PURPOSE: This study assessed the in vitro effect of SOL on promastigotes and Leishmania amazonensis-infected macrophages, as well its microbicide and immunomodulatory mechanisms. METHODS: SOL was isolated from the roots of S. chilensis, 98% purity, and identified by chromatographic methods, and the effect of SOL on leishmanicidal activity against promastigotes in vitro, SOL-induced cytotoxicity in THP-1, J774 cells, sheep erythrocytes, and L. amazonensis-infected J774 macrophages, and the mechanisms of death involved in this action were evaluated. RESULTS: In silico predictions showed good drug-likeness potential for SOL with high oral bioavailability and intestinal absorption. SOL treatment (10-160 µM) inhibited promastigote proliferation 24, 48, and 72 h after treatment. After 24 h of treatment, SOL at the IC50 (34.5 µM) and 2 × the IC50 (69 µM) induced several morphological and ultrastructural changes in promastigotes, altered the cell cycle and cellular volume, increased phosphatidylserine exposure on the cell surface, induced the loss of plasma membrane integrity, increased the reactive oxygen species (ROS) level, induced loss of mitochondrial integrity (characterized by an apoptosis-like process), and increased the number of lipid droplets and autophagic vacuoles. Additionally, SOL induced low cytotoxicity in J774 murine macrophages (CC50 of 1587 µM), THP-1 human monocytes (CC50 of 1321 µM), and sheep erythrocytes. SOL treatment reduced the percentage of L. amazonensis-infected macrophages and the number of amastigotes per macrophage (IC50 9.5 µM), reduced TNF-α production and increased IL-12p70, ROS and nitric oxide (NO) levels. CONCLUSION: SOL showed in vitro leishmanicidal effects against the promastigotes by apoptosis-like mechanism and amastigotes by reducing TNF-α and increasing IL-12p70, ROS, and NO levels, suggesting their potential as a candidate for use in further studies on the design of antileishmanial drugs.
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Apoptosis/efectos de los fármacos , Furanos/farmacología , Leishmania/efectos de los fármacos , Macrófagos/efectos de los fármacos , Naftalenos/farmacología , Animales , Antiprotozoarios/farmacología , Línea Celular , Humanos , Macrófagos/parasitología , Ratones , Ratones Endogámicos BALB C , Mitocondrias/metabolismo , Mitocondrias/patología , Óxido Nítrico/metabolismo , Fosfatidilserinas/metabolismo , Raíces de Plantas/química , Especies Reactivas de Oxígeno/metabolismo , Ovinos , Solidago/química , Células THP-1RESUMEN
Plasma membrane tubulin is an endogenous regulator of P-ATPases and the unusual accumulation of tubulin in the erythrocyte membrane results in a partial inhibition of some their activities, causing hemorheological disorders like reduced cell deformability and osmotic resistance. These disorders are of particular interest in hypertension and diabetes, where the abnormal increase in membrane tubulin may be related to the disease development. Phosphatidylserine (PS) is more exposed on the membrane of diabetic erythrocytes than in healthy cells. In most cells, PS is transported from the exoplasmic to the cytoplasmic leaflet of the membrane by lipid flippases. Here, we report that PS is more exposed in erythrocytes from both hypertensive and diabetic patients than in healthy erythrocytes, which could be attributed to the inhibition of flippase activity by tubulin. This is supported by: (i) the translocation rate of a fluorescent PS analog in hypertensive and diabetic erythrocytes was slower than in healthy cells, (ii) the pharmacological variation of membrane tubulin in erythrocytes and K562 cells was linked to changes in PS translocation and (iii) the P-ATPase-dependent PS translocation in inside-out vesicles (IOVs) from human erythrocytes was inhibited by tubulin. These results suggest that tubulin regulates flippase activity and hence, the membrane phospholipid asymmetry.
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Adenosina Trifosfatasas/antagonistas & inhibidores , Diabetes Mellitus/patología , Eritrocitos/metabolismo , Hipertensión/patología , Fosfatidilserinas/metabolismo , Tubulina (Proteína)/metabolismo , Adenosina Trifosfatasas/metabolismo , Adulto , Estudios de Casos y Controles , Diabetes Mellitus/metabolismo , Femenino , Humanos , Hipertensión/metabolismo , Masculino , Persona de Mediana EdadRESUMEN
The rapid ability of SARS-CoV-2 to spread among humans, along with the clinical complications of coronavirus disease 2019-COVID-19, have represented a significant challenge to the health management systems worldwide. The acute inflammation and coagulation abnormalities appear as the main causes for thousands of deaths worldwide. The intense inflammatory response could be involved with the formation of thrombi. For instance, the presence of uncleaved large multimers of von Willebrand (vWF), due to low ADAMTS13 activity in plasma could be explained by the inhibitory action of pro-inflammatory molecules such as IL-1ß and C reactive protein. In addition, the damage to endothelial cells after viral infection and/or activation of endothelium by pro-inflammatory cytokines, such as IL-1ß, IL-6, IFN-γ, IL-8, and TNF-α induces platelets and monocyte aggregation in the vascular wall and expression of tissue factor (TF). The TF expression may culminate in the formation of thrombi, and activation of cascade by the extrinsic pathway by association with factor VII. In this scenario, the phosphatidylserine-PtdSer exposure on the outer leaflet of the cell membrane as consequence of viral infection emerges as another possible underlying mechanism to acute immune inflammatory response and activation of coagulation cascade. The PtdSer exposure may be an important mechanism related to ADAM17-mediated ACE2, TNF-α, EGFR and IL-6R shedding, and the activation of TF on the surface of infected endothelial cells. In this review, we address the underlying mechanisms involved in the pathophysiology of inflammation and coagulation abnormalities. Moreover, we introduce key biochemical and pathophysiological concepts that support the possible participation of PtdSer exposure on the outer side of the SARS-CoV-2 infected cells membrane, in the pathophysiology of COVID-19. Video Abstract.
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COVID-19/genética , Inflamación/genética , Fosfatidilserinas/genética , Trombosis/genética , Proteína ADAM17/genética , Proteína ADAMTS13/genética , COVID-19/complicaciones , COVID-19/patología , COVID-19/virología , Células Endoteliales/virología , Humanos , Inflamación/complicaciones , Inflamación/virología , Fosfatidilserinas/metabolismo , Receptores de Interleucina-6/genética , SARS-CoV-2/patogenicidad , Trombosis/patología , Trombosis/virología , Factor de von Willebrand/genéticaRESUMEN
In the context of the cancer-inflammation relationship and the use of natural products as potential antitumor and anti-inflammatory agents, the alkaloid-enriched fraction of Boehmeriacaudata (BcAEF) aerial parts was evaluated. In vitro antiproliferative studies with human tumor cell lines showed high activity at low concentrations. Further investigation on NCI-H460 cells showed an irreversible effect on cell proliferation, with cell cycle arrest at G2/M phase and programmed cell death induction. Molecular docking studies of four alkaloids identified in BcAEF with colchicine's binding site on ß-tubulin were performed, suggesting (-)-C (15R)-hydroxycryptopleurine as the main inductor of the observed mitotic death. In vivo studies showed that BcAEF was able to reduce Ehrlich tumor volume progression by 30 to 40%. Checking myeloperoxidase activity, BcAEF reduced neutrophils migration towards the tumor. The in vivo anti-inflammatory activity was evaluated by chemically induced edema models. In croton oil-induced ear edema and carrageenan (CG)-induced paw edema models, BcAEF reduced edema around 70 to 80% together with inhibition of activation and/or migration of neutrophils to the inflammatory area. All together the results presented herein show BcAEF as a potent antitumor agent combining antiproliferative and anti-inflammatory properties, which could be further explored in (pre)clinical studies.
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Alcaloides/química , Antiinflamatorios/farmacología , Antineoplásicos/farmacología , Boehmeria/química , Simulación por Computador , Extractos Vegetales/farmacología , Animales , Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Clonales , Modelos Animales de Enfermedad , Oído/patología , Edema/patología , Activación Enzimática/efectos de los fármacos , Exocitosis , Humanos , Simulación del Acoplamiento Molecular , Paclitaxel/farmacología , Peroxidasa/metabolismo , Fosfatidilserinas/metabolismo , Estándares de Referencia , Pruebas de Toxicidad AgudaRESUMEN
Bone biomineralization is an exquisite process by which a hierarchically organized mineral matrix is formed. Growing evidence has uncovered the involvement of one class of extracellular vesicles, named matrix vesicles (MVs), in the formation and delivery of the first mineral nuclei to direct collagen mineralization. MVs are released by mineralization-competent cells equipped with a specific biochemical machinery to initiate mineral formation. However, little is known about the mechanisms by which MVs can trigger this process. Here, we present a combination of in situ investigations and ex vivo analysis of MVs extracted from growing-femurs of chicken embryos to investigate the role played by phosphatidylserine (PS) in the formation of mineral nuclei. By using self-assembled Langmuir monolayers, we reconstructed the nucleation core - a PS-enriched motif thought to trigger mineral formation in the lumen of MVs. In situ infrared spectroscopy of Langmuir monolayers and ex situ analysis by transmission electron microscopy evidenced that mineralization was achieved in supersaturated solutions only when PS was present. PS nucleated amorphous calcium phosphate that converted into biomimetic apatite. By using monolayers containing lipids extracted from native MVs, mineral formation was also evidenced in a manner that resembles the artificial PS-enriched monolayers. PS-enrichment in lipid monolayers creates nanodomains for local increase of supersaturation, leading to the nucleation of ACP at the interface through a multistep process. We posited that PS-mediated nucleation could be a predominant mechanism to produce the very first mineral nuclei during MV-driven bone/cartilage biomineralization.
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Biomineralización/fisiología , Fosfatos de Calcio/metabolismo , Lípidos/fisiología , Fosfatidilserinas/metabolismo , Animales , Apatitas/metabolismo , Biomimética/métodos , Calcificación Fisiológica/fisiología , Calcio/metabolismo , Cartílago/metabolismo , Pollos , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Vesículas Extracelulares/metabolismo , Fémur/metabolismo , Microscopía Electrónica de Transmisión/métodosRESUMEN
The P2X7 receptor is a ligand-gated, cation-selective channel whose main physiological ligand is ATP. P2X7 receptor activation may also be triggered by ARTC2.2-dependent ADP ribosylation in the presence of extracellular NAD. Upon activation, this receptor induces several responses, including the influx of calcium and sodium ions, phosphatidylserine externalization, the formation of a non-selective membrane pore, and ultimately cell death. P2X7 receptor activation depends on the availability of extracellular nucleotides, whose concentrations are regulated by the action of extracellular nucleotidases such as CD39 and CD38. The P2X7 receptor has been extensively studied in the context of the immune response, and it has been reported to be involved in inflammasome activation, cytokine production, and the migration of different innate immune cells in response to ATP. In adaptive immune responses, the P2X7 receptor has been linked to T cell activation, differentiation, and apoptosis induction. In this review, we will discuss the evidence of the role of the P2X7 receptor on T cell differentiation and in the control of T cell responses in inflammatory conditions.
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Receptores Purinérgicos P2X7/fisiología , Subgrupos de Linfocitos T/inmunología , ADP-Ribosil Ciclasa 1/fisiología , Adenosina Trifosfato/fisiología , Animales , Antígenos CD/fisiología , Apoptosis/fisiología , Apirasa/fisiología , Diferenciación Celular/fisiología , Células Dendríticas/citología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Humanos , Inflamasomas/metabolismo , Activación del Canal Iónico/fisiología , Activación de Linfocitos/fisiología , Ratones , Nucleótidos/metabolismo , Fosfatidilserinas/metabolismo , Ratas , Receptores Purinérgicos P2X7/química , Receptores Purinérgicos P2X7/efectos de los fármacos , Receptores Purinérgicos P2X7/genética , Transducción de Señal/fisiología , Relación Estructura-Actividad , Subgrupos de Linfocitos T/metabolismoRESUMEN
OBJECTIVE: This study aimed to assess prospectively the role of anti-ß2-glycoprotein I domain I antibody (aß2GPI-DI) and the Global Antiphospholipid Syndrome Score (GAPSS) in identifying antiphospholipid syndrome (APS) patients at higher risk of a new event. METHODS: Thrombotic APS patients were followed from May 2013 to July 2017. At baseline, we measured lupus anticoagulant, IgG/IgM anticardiolipin, anti-ß2-glycoprotein I, antiphosphatidylserine-prothrombin (aPS/PT) and IgG aß2GPI-DI, and calculated GAPSS for each patient. RESULTS: A total of 44 patients (age 43 ± 10 years, 89% female, 73% primary APS) were followed for 39 months (range 9-46 months). Four new thromboses occurred, two of them after vitamin K antagonist interruption. Recurrent patients presented higher GAPSS (median 20) and were triple and aß2GPI-DI positive; non-recurrent patients had lower GAPSS (median 10.5, range 0-20) and lower ratio of triple (33%) and aß2GPI-DI positivities (38%). aß2GPI-DI was associated with higher GAPSS (median 19 vs. 7, p < 0.001; Pearson correlation 0.82, p < 0.001) and had a greater proportion of triple (83% vs. 4%, p < 0.001) and aPS/PT positivity (94% vs. 50%, p = 0.002). CONCLUSION: Our data show a significant correlation between a validated risk score such as GAPSS and the novel antiphospholipid antibody aß2GPI-DI. Future studies are needed. However, one could speculate a role of aß2GPI-DI as a risk-stratifying tool for thrombotic events in APS.