RESUMO
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.
Assuntos
Membrana Celular , Pró-Fármacos , Uridina , Pró-Fármacos/química , Pró-Fármacos/farmacologia , Pró-Fármacos/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Uridina/química , Uridina/farmacologia , Fosfatidilserinas/química , Termodinâmica , Propriedades de Superfície , Viscosidade , ElasticidadeRESUMO
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.
Assuntos
Anexinas , Lipossomos , Anexina A5/química , Anexina A5/metabolismo , Fosfatidilserinas/química , Fosfatidilserinas/metabolismo , Biomimética , Proteômica , Cálcio/metabolismoRESUMO
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.
Assuntos
Preservação do Sêmen , Sêmen , Masculino , Animais , Bovinos , Congelamento , Antioxidantes/farmacologia , Fosfatidilserinas , Motilidade dos Espermatozoides , Preservação do Sêmen/veterinária , Crioprotetores/farmacologia , Espermatozoides , Análise do Sêmen/veterinária , Criopreservação/veterinária , Suplementos NutricionaisRESUMO
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.
Assuntos
Ixodidae , Febre Maculosa das Montanhas Rochosas , Carrapatos , Animais , Humanos , Coelhos , Carrapatos/microbiologia , Amblyomma , Caspase 3/metabolismo , Ixodidae/genética , Proteínas Inibidoras de Apoptose/metabolismo , Células Endoteliais , Fosfatidilserinas/metabolismo , Rickettsia rickettsii/fisiologia , BrasilRESUMO
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.
Assuntos
Antiprotozoários , Produtos Biológicos , Leishmania infantum , Camundongos , Animais , Terpenos/farmacologia , Fosfatidilserinas , Propídio , Camundongos Endogâmicos BALB C , Antiprotozoários/toxicidade , Antiprotozoários/uso terapêutico , Produtos Biológicos/farmacologiaRESUMO
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.
Assuntos
Toxinas Bacterianas , Clostridioides difficile , Conexinas , Proteínas do Tecido Nervoso , Receptores Purinérgicos P2X7 , Animais , Anexinas , Toxinas Bacterianas/metabolismo , Caspase 3/metabolismo , Morte Celular , Conexinas/genética , Conexinas/metabolismo , Mediadores da Inflamação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neuroglia/metabolismo , Fosfatidilserinas , Receptores Purinérgicos P2X7/genéticaRESUMO
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.
Assuntos
Anemia Falciforme , Losartan , Acrilatos , Angiotensina II/metabolismo , Angiotensina II/farmacologia , Anexina A5 , Eritrócitos/metabolismo , Hemoglobina Falciforme/metabolismo , Humanos , Imidazóis , Losartan/farmacologia , Fosfatidilserinas , Polimerização , Receptor Tipo 1 de Angiotensina/metabolismo , TiofenosRESUMO
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.
Assuntos
Anexina A6 , Calcinose , 1,2-Dipalmitoilfosfatidilcolina/química , Anexina A6/metabolismo , Colágeno/metabolismo , Humanos , Fosfatos/metabolismo , Fosfatidilserinas/química , ProteolipídeosRESUMO
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.
Assuntos
Anestesiologistas , Adulto , Azidas , Humanos , Recém-Nascido , Fosfatidilserinas , Estudos Prospectivos , Reprodutibilidade dos Testes , AutorrelatoRESUMO
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.