RESUMEN
Eicosanoids are a family of bioactive lipids, including derivatives of the ubiquitous fatty acid arachidonic acid (AA). The intimate involvement of eicosanoids in inflammation motivates the development of predictive in silico models for a systems-level exploration of disease mechanisms, drug development and replacement of animal models. Using an ensemble modelling strategy, we developed a computational model of the AA cascade. This approach allows the visualisation of plausible and thermodynamically feasible predictions, overcoming the limitations of fixed-parameter modelling. A quality scoring method was developed to quantify the accuracy of ensemble predictions relative to experimental data, measuring the overall uncertainty of the process. Monte Carlo ensemble modelling was used to quantify the prediction confidence levels. Model applicability was demonstrated using mass spectrometry mediator lipidomics to measure eicosanoids produced by HaCaT epidermal keratinocytes and 46BR.1N dermal fibroblasts, treated with stimuli (calcium ionophore A23187), (ultraviolet radiation, adenosine triphosphate) and a cyclooxygenase inhibitor (indomethacin). Experimentation and predictions were in good qualitative agreement, demonstrating the ability of the model to be adapted to cell types exhibiting differences in AA release and enzyme concentration profiles. The quantitative agreement between experimental and predicted outputs could be improved by expanding network topology to include additional reactions. Overall, our approach generated an adaptable, tuneable ensemble model of the AA cascade that can be tailored to represent different cell types and demonstrated that the integration of in silico and in vitro methods can facilitate a greater understanding of complex biological networks such as the AA cascade.
Asunto(s)
Ácido Araquidónico , Simulación por Computador , Queratinocitos , Ácido Araquidónico/metabolismo , Humanos , Queratinocitos/metabolismo , Lipidómica/métodos , Método de Montecarlo , Eicosanoides/metabolismo , Modelos Biológicos , Fibroblastos/metabolismo , Línea Celular , Calcimicina/farmacologíaRESUMEN
Protein glycosylation is a post-translational modification involved in wide range of biological processes. In mammalian spermatozoa this modification has been identified in numerous proteins, and membrane glycoproteins are involved in the fertilization process. The objective of the present study was to identify changes in protein glycosylation after acrosome reaction (AR) induction using the 4-Br-A23187 ionophore. Our results showed that treatment with 10 µM of 4-Br-A23187 for 20 min significantly increased the percentage of live acrosome-reacted spermatozoa compared to the control (69.8 ± 0.8 vs. 6.4 ± 0.5; mean % ± SEM, respectively). Also, we observed an increase in 32 kDa tyrosine-phosphorylated protein (p32) and a decrease in serine/threonine phosphorylation of the protein kinase A substrates (phospho-PKA-substrates) after ionophore treatment. Furthermore, changes in glycosylated proteins following AR induction were analyzed using different HRP-conjugated lectins (GNA, DSA, and SNA), revealing changes in mannose and sialic acid residues. Proteomic analysis of isolated proteins using GNA lectin revealed that 50 proteins exhibited significantly different abundance (q-value < 0.01). Subsequent analysis using Uniprot database identified 39 downregulated and 11 upregulated proteins in the presence of 4-Br-A23187. Notably, six of these proteins were classified as transmembrane proteins, namely LRRC37A/B like protein 1 C-terminal domain-containing protein, Membrane metalloendopeptidase like 1, VWFA domain-containing protein, Syndecan, Membrane spanning 4-domains A14 and Serine protease 54. This study shows a novel protocol to induce acrosome reaction in boar spermatozoa and identifies new transmembrane proteins containing mannose residues. Further work is needed to elucidate the role of these proteins in sperm-oocyte fusion.
Asunto(s)
Reacción Acrosómica , Calcimicina , Espermatozoides , Animales , Masculino , Reacción Acrosómica/efectos de los fármacos , Porcinos , Espermatozoides/metabolismo , Espermatozoides/efectos de los fármacos , Calcimicina/farmacología , Glicoproteínas/metabolismo , Glicosilación , Proteoma , Ionóforos de Calcio/farmacologíaRESUMEN
This study explores Neutrophil Extracellular Trap (NET) formation in equine neutrophils, which is crucial for eliminating infections and is implicated in various equine inflammatory diseases. We investigated the molecular pathways involved in NET release by equine neutrophils in response to stimuli. We use PMA, A23187, LPS, PAF, OZ, and cytokines, observing NET release in response to PMA, PAF, and A23187. In contrast, LPS, OZ, and the cytokines tested did not induce DNA release or did not consistently induce citrullination of histone 4. Peptidyl-arginine deiminase inhibition completely halted NET release, while NADPH oxidase and mitochondrial reactive oxygen species only played a role in PMA-induced NETs. Neutrophil elastase inhibition modestly affected PAF-induced NET liberation but not in PMA or A23187-induced NET, while myeloperoxidase did not contribute to NET release. We expect to provide a foundation for future investigations into the role of NETs in equine health and disease and the search for potential therapeutic targets.
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Trampas Extracelulares , Neutrófilos , Animales , Caballos , Neutrófilos/metabolismo , Trampas Extracelulares/metabolismo , Calcimicina/metabolismo , Lipopolisacáridos/metabolismo , Citocinas/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
UV-B radiation induces sunburn, and neutrophils are pivotal in this inflammation. In this study, we examined the potential involvement of neutrophil extracellular traps (NETs) in ultraviolet B (UVB)-induced skin inflammation, correlating the skin inflammation-mitigating effects of Hochu-ekki-to on UV-B irradiation and NETs. To elucidate NET distribution in the dorsal skin, male ICR mice, exposed to UVB irradiation, were immunohistologically analyzed to detect citrullinated histone H3 (citH3) and peptidylarginine deiminase 4 (PAD4). Reactive oxygen species (ROS) production in the bloodstream was analyzed. To establish the involvement of NET-released DNA in this inflammatory response, mice were UV-B irradiated following the intraperitoneal administration of DNase I. In vitro experiments were performed to scrutinize the impact of Hochu-ekki-to on A23187-induced NETs in neutrophil-like HL-60 cells. UV-B irradiation induced dorsal skin inflammation, coinciding with a significant increase in citH3 and PAD4 expression. Administration of DNase I attenuated UV-B-induced skin inflammation, whereas Hochu-ekki-to administration considerably suppressed the inflammation, correlating with diminished levels of citH3 and PAD4 in the dorsal skin. UV-B irradiation conspicuously augmented ROS and hydrogen peroxide (H2O2) production in the blood. Hochu-ekki-to significantly inhibited ROS and H2O2 generation. In vitro experiments demonstrated that Hochu-ekki-to notably inhibited A23187-induced NETs in differentiated neutrophil-like cells. Hence, NETs have been implicated in UV-B-induced skin inflammation, and their inhibition reduces cutaneous inflammation. Additionally, Hochu-ekki-to mitigated skin inflammation by impeding neutrophil infiltration and NETs in the dorsal skin of mice.
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Desoxirribonucleasa I , Medicamentos Herbarios Chinos , Trampas Extracelulares , Rayos Ultravioleta , Animales , Masculino , Ratones , Calcimicina/farmacología , Desoxirribonucleasa I/farmacología , Desoxirribonucleasa I/metabolismo , Trampas Extracelulares/efectos de los fármacos , Trampas Extracelulares/efectos de la radiación , Histonas/metabolismo , Peróxido de Hidrógeno/metabolismo , Inflamación/metabolismo , Ratones Endogámicos ICR , Neutrófilos/metabolismo , Desiminasas de la Arginina Proteica/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Rayos Ultravioleta/efectos adversosRESUMEN
A novel antibiotic biosynthetic precursor of cezomycin, named precezomycin (1), was isolated from culture broth of actinomycete Kitasatospora putterlickiae 10-13. The planar structure was determined by 1D/2D NMR and HR(ESI)MS data analyses, and the absolute configurations were established by TDDFT calculation of ECD spectra. Precezomycin (1) exhibited moderate antibacterial activity against gram-positive bacteria including Staphylococcus aureus and Bacillus subtilis. The discovery of 1 extends the natural product family of cezomycin and provides a new insight into understanding the biosynthetic process of these polyether ionophore metabolites.
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Actinobacteria , Calcimicina/análogos & derivados , Streptomyces , Streptomycetaceae , Streptomyces/metabolismo , Antibacterianos/química , Pruebas de Sensibilidad Microbiana , Estructura MolecularRESUMEN
BACKGROUND: Equine spermatozoa appear to differ from spermatozoa of other species in using oxidative phosphorylation preferentially over glycolysis. However, there is little information regarding effects of different energy sources on measured parameters in equine spermatozoa. OBJECTIVE: To determine the effect of three individual energy substrates, glucose, pyruvate, and lactate, on motion characteristics, membrane integrity, and acrosomal status of stallion spermatozoa. MATERIALS AND METHODS: Freshly ejaculated stallion spermatozoa were incubated with combinations of glucose (5 mm), pyruvate (10 mm), and lactate (10 mm) for 0.5 to 4 h. Response to calcium ionophore A23187 (5 µm) was used to evaluate capacitation status. Motility was evaluated using computer-assisted sperm analysis, and plasma membrane and acrosomal integrity were evaluated by flow cytometry. RESULTS: Incubation with lactate alone for 2 h increased acrosomal sensitivity to A23187. Notably, incubation with lactate alone for 4 h induced a significant spontaneous increase in acrosome-reacted, membrane-intact (viable) spermatozoa, to approximately 50% of the live population, whereas no increase was seen with incubation in glucose or pyruvate alone. This acrosomal effect was observed in spermatozoa incubated at physiological pH as well as under alkaline conditions (medium pH approximately 8.5). Sperm motility declined concomitantly with the increase in acrosome-reacted spermatozoa. Sperm motility was significantly higher in pyruvate-only medium than in glucose or lactate. The addition of pyruvate to lactate-containing medium increased sperm motility but reduced the proportion of live acrosome-reacted spermatozoa in a dose-dependent fashion. DISCUSSION: This is the first study to demonstrate that incubation with a specific energy substrate, lactate, is associated with spontaneous acrosome reaction in spermatozoa. The proportion of live, acrosome-reacted spermatozoa obtained is among the highest reported for equine spermatozoa. CONCLUSION: These findings highlight the delicate control of key sperm functions, and may serve as a basis to increase our understanding of stallion sperm physiology.
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Reacción Acrosómica , Ácido Láctico , Masculino , Animales , Caballos , Reacción Acrosómica/fisiología , Ácido Láctico/metabolismo , Calcimicina/farmacología , Semen , Motilidad Espermática , Espermatozoides/metabolismo , Acrosoma , Piruvatos/metabolismo , Piruvatos/farmacología , Glucosa/metabolismo , Capacitación EspermáticaRESUMEN
The goal of the present work was to develop novel ß-substituted-α-halomethyl acrylates from a methodology in an aqueous phase and to evaluate their bioactivity as potential inhibitors of mast cell activation. Eleven ß-substituted-α-halomethyl acrylates were synthesized through a modified Horner-Wadsworth-Emmons reaction. Compound 48/80 and the calcium ionophore A23187 stimulated the release of ß-hexosaminidase from mast cells. The effect induced by compound 48/80 was inhibited by compound 5 (320 µM) and compound 9 (160 and 320 µM) without causing cytotoxic effects. The effect induced by A23187 was inhibited by compound 5 (40, 80, 160, and 320 µM) without affecting cell viability. The inhibitory effects exhibited by compounds 5 and 9 were more potent than those of the reference compound sodium cromoglycate at the same concentrations. The biochemical results were consistent with the morphological findings obtained by light and transmission electron microscopy. This study reports, for the first time, that the new synthetic compounds methyl (Z)- 2-bromo-3-(furan-3-yl)acrylate (compound 5) and methyl (E)- 2-bromo-3-(3-bromophenyl)acrylate (compound 9) strongly inhibit mast cell degranulation, without affecting cell viability. The implications of these results are relevant as a basis for developing new anti-inflammatory and mast cell stabilizing drugs.
Asunto(s)
Degranulación de la Célula , Mastocitos , Calcimicina/farmacología , Acrilatos/farmacología , p-Metoxi-N-metilfenetilamina/farmacologíaRESUMEN
BACKGROUND: Calcimycin (A23187) is a polyether antibiotic and divalent cation ionophore, extracted from Streptomyces chartrecensis. With wide variety of antimicrobial activities, it also exhibits cytotoxicity of tumor cells. Calcimycin exhibit therapeutic potential against tumor cell growth; however, the molecular mechanism remains to be fully elucidated. Present study explores the mechanism of calcimycin-induced apoptosis cancer cell lines. METHODS: Apoptotic induction in a dose-dependent manner were recorded with MTT assays, Phase contrast imaging, wound healing assay, fluorescence imaging by DAPI and AO/EB staining and FACS using cell line model. Mitochondrial potential was analyzed by TMRM assay as Ca2+ signaling is well known to be influenced and synchronized by mitochondria also. RESULTS: Calcimycin induces apoptosis in dose dependent manner, also accompanied by increased intracellular calcium-level and expression of purinergic receptor-P2RX4, a ligand-gated ion channel. CONCLUSION: Calcimycin tends to increase the intracellular calcium level, mRNA expression of ATP receptor P2RX4, and phosphorylation of p38. Blocking of either intracellular calcium by BAPTA-AM, P2RX4 expression by antagonist 5-BDBD, and phospho-p38 by SB203580, abrogated the apoptotic activity of calcimycin. GENERAL SIGNIFICANCE: Taken together, these results show that calcimycin induces apoptosis in P2RX4 and ATP mediated intracellular Ca2+ and p38 MAPK mediated pathway in both the cancer cell lines. This study explored a new mode of action for calcimycin in cancer that could be potentially employed in future studies for cancer therapeutic research. This study disentangles that the calcimycin-induced apoptotic cell death is P2RX4 and ATP involved, intracellular Ca2+ and p38 MAPK mediated pathway.
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Apoptosis , Calcimicina , Calcio , Receptores Purinérgicos P2X4 , Células MCF-7 , Línea Celular Tumoral , Humanos , Calcimicina/farmacología , Apoptosis/efectos de los fármacos , Calcio/metabolismo , Receptores Purinérgicos P2X4/metabolismo , Espacio Intracelular/metabolismo , Proliferación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Oxygen-glucose deprivation (OGD) is a critical model for studying hypoxic-ischemic cerebrovascular disease in vitro. This paper is to investigate the protection of OGD-induced cellular damage and inflammatory responses by OGD preconditioning in vitro, to provide a theoretical basis for OGD preconditioning to improve the prevention and prognosis of ischemic stroke. OGD or OGD preconditioning model was established by culturing the PC12 cell line in vitro, followed by further adding A23187 (calcium ion carrier) or CsA (calcium ion antagonist). Cell viability was detected by MTT, apoptosis by Hoechst 33,258 staining, the levels of TNF-α and IL-1ß mRNA by RT-qPCR and ELISA, and the levels of CaN, NFAT, COX-2 by RT-qPCR and Western blot. Cell viability was decreased, and apoptosis, inflammatory cytokines, and CaN, NFAT, and COX-2 levels were notably increased upon OGD, while OGD pretreatment significantly increased cell viability and decreased apoptosis, inflammation, and the Ca2+/CaN/NFAT pathway. Treatment with A23187 decreased cell viability, promoted apoptosis, and significantly increased TNF-α, IL-1ß, CaN, NFAT, and COX-2 levels, while CsA treatment reduced the opposite results. In vitro OGD preconditioning mediates the Ca2+/CaN/NFAT pathway to protect against OGD-induced cellular damage and inflammatory responses.
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Precondicionamiento Isquémico , Oxígeno , Ratas , Animales , Oxígeno/metabolismo , Calcio , Factor de Necrosis Tumoral alfa , Glucosa , Calcimicina , Ciclooxigenasa 2 , Apoptosis , Células PC12 , Supervivencia CelularRESUMEN
Intestinal ischemia-reperfusion (IIR) injury is associated with inflammation and oxidative stress, yet its precise mechanisms remain not fully understood. IIR injury is closely linked to the gut microbiota and its metabolites. The anti-inflammatory and antioxidant effects of Lactiplantibacillus plantarum are specific to IIR. In our study, we conducted a 30-day pre-treatment of SD rats with both a standard strain of Lactiplantibacillus plantarum and Lactiplantibacillus plantarum GL001. After a 7-day cessation of treatment, we induced an IIR injury model to investigate the mechanisms by which Lactiplantibacillus plantarum alleviates IIR damage. The results demonstrate that Lactiplantibacillus plantarum effectively mitigates the inflammatory and oxidative stress damage induced by IIR. Lactiplantibacillus plantarum GL001 can improve the gut microbiota by reducing the abundance of harmful bacteria and increasing the abundance of beneficial bacteria. In IIR intestinal tissue, the levels of secondary bile acids are elevated. The content of the bacterial metabolite Calcimycin increases. Annotations of metabolic pathways suggest that Lactiplantibacillus plantarum GL001 can alleviate IIR damage by modulating calcium-phosphorus homeostasis through the regulation of parathyroid hormone synthesis, secretion, and action. Microbiota-metabolite correlation analysis reveals a significant negative correlation between calcimycin and Lactonacillus and a significant positive correlation between calcimycin and Shigella. There is also a significant positive correlation between calcimycin and secondary bile acids. Lactiplantibacillus plantarum GL001 can alleviate oxidative damage induced by IIR through improvements in gut microbiota and intestinal tissue metabolism.
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Estrés Oxidativo , Daño por Reperfusión , Ratas , Animales , Calcimicina/farmacología , Ratas Sprague-Dawley , Daño por Reperfusión/metabolismo , Bacterias , Ácidos y Sales BiliaresRESUMEN
Confocal microscopy and fluorescence staining of cellular structures are commonly used to study neutrophil activation and NETosis. However, they do not reveal the specific characteristics of the neutrophil membrane surface, its nanostructure, and morphology. The aim of this study was to reveal the topography and nanosurface characteristics of neutrophils during activation and NETosis using atomic force microscopy (AFM). We showed the main stages of neutrophil activation and NETosis, which include control cell spreading, cell fragment formation, fusion of nuclear segments, membrane disruption, release of neutrophil extracellular traps (NETs), and final cell disintegration. Changes in neutrophil membrane nanosurface parameters during activation and NETosis were quantified. It was shown that with increasing activation time there was a decrease in the spectral intensity of the spatial periods. Exposure to the activator A23187 resulted in an increase in the number and average size of cell fragments over time. Exposure to the activators A23187 and PMA (phorbol 12-myristate 13-acetate) caused the same pattern of cell transformation from spherical cells with segmented nuclei to disrupted cells with NET release. A23187 induced NETosis earlier than PMA, but PMA resulted in more cells with NETosis at the end of the specified time interval (180 min). In our study, we used AFM as the main research tool. Confocal laser-scanning microscopy (CLSM) images are provided for identification and detailed analysis of the phenomena studied. In this way, we exploited the advantages of both techniques.
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Trampas Extracelulares , Neutrófilos , Calcimicina , Microscopía de Fuerza Atómica , Núcleo Celular , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Acrosome is inextricably related to membranous organelles. The origin of acrosome is still controversial, one reason is that limited articles were reported about the proteomic analysis of the acrosome. Mitochondrial proteins were found exist in the acrosome, nevertheless, only limited attention has been paid to the function of mitochondrial proteins in the acrosome formation. Eriocheir sinensis sperm has a large acrosome, which makes it an ideal model to study acrosome formation. Here, we firstly compared the rate of acrosome reaction induced by the calcium ionophore A23187 and ionomycin. The rate of acrosome reaction induced by ionomycin is higher (95.8%) than A23187 (58.7%). Morphological changes were observed using light, confocal and transmission electron microscopy. Further more, proteins released during the acrosome reaction as induced by ionomycin were collected for LC-MS/MS analysis. A total of 945 proteins, including malate dehydrogenase (MDH) and voltage-dependent anion channel 3 (VDAC3), were identified in the acrosomal released proteome. The number of proteins from mitochondria (17.57%) was higher compared with endoplasmic reituculum (1.59%) and lysosomes (1.8%). To investigate the functions of target mitochondrial proteins during spermatogenesis, poly-antibodies of MDH in E. sinensis were prepared. The characteristics, further analyzed using immunofluorescence, of two mitochondrial proteins during acrosome formation showed that MDH and VDAC3 were independently involved in the formation of acrosomal membrane. These findings illustrate the acrosomal released proteome and provide important data resource for understanding the relationship between mitochondria and the acrosome in Decapoda crustacean.
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Malato Deshidrogenasa , Proteoma , Masculino , Humanos , Acrosoma , Calcimicina , Cromatografía Liquida , Ionomicina , Proteómica , Semen , Espectrometría de Masas en Tándem , Espermatozoides , Espermatogénesis , Mitocondrias , Proteínas Mitocondriales , Canales Aniónicos Dependientes del Voltaje , LisosomasRESUMEN
Before NETs are released, the neutrophil undergoes structural changes. First, it flattens, accompanied by a change in cell shape and rearrangement of the cytoskeleton. Then, nuclear swelling begins, which ends with the ejection of NETs into the extracellular space. We used widefield and confocal fluorescence microscopy to register morphological and structural changes in neutrophils during activation and NETosis. Different types of activators were used, such as NOX-dependent PMA and calcium ionophore A23187. The measurements were performed in a series of sequential stages. In the first stage (30 s after addition of activators and immediately after stimulation of neutrophils), the response of neutrophils to A23187 and PMA exposure was studied. Subsequently, the characteristics of neutrophils in different phases of activation were examined over a longer period of time (30, 60, 120, 180, and 240 min). The specific features of NETosis development were analyzed separately. During the first 30 s, neutrophils appeared to be heterogeneous in shape and structure of the actin cytoskeleton. Characteristic cell shapes included 30â³ type 1 cells, similar in shape to the control, with F-actin concentrated in the center of the cytoplasm, and 30â³ type 2 cells, which had flattened (spread) shapes with increased frontal dimensions and F-actin distributed throughout the cell. Later, the development of nuclear swelling, the corresponding changes in neutrophil membranes, and NET release into the extracellular space were evaluated. The conditions determining the initiation of chromatin ejection and two characteristic types of decondensed chromatin ejection were revealed. The results obtained contribute to a better understanding of the biophysical mechanisms of neutrophil activation and NETosis development.
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Trampas Extracelulares , Neutrófilos , Neutrófilos/metabolismo , Calcimicina/farmacología , Actinas/metabolismo , Trampas Extracelulares/metabolismo , Cromatina/metabolismoRESUMEN
Hypercholesterolemia is a major complication of arteriosclerosis. Mast cells in arteriosclerosis plaques induce inflammatory reactions and promote arterial sclerosis. In this study, we evaluated the pharmacological effects of simvastatin (SV)-3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitors on the degranulation of rat basophilic leukemia (RBL)-2H3 cells, which are commonly used as mast cell models. SV significantly decreased the degranulation induced by three types of stimulation: antigen antibody reaction (Ag-Ab), thapsigargin (Tg) serosal endoplasmic reticulum calcium ATPase (SERCA) inhibitor, and A23187 calcium ionophore. SV had a stronger inhibitory effect on degranulation induced by Ag-Ab stimulation than the other two stimulations. However, SV did not inhibit increase of intracellular Ca2+ concentrations. Mevalonate or geranylgeraniol co-treatment with SV completely prevented the inhibitory effect of SV on the degranulation induced by these stimulations. Immunoblotting results showed that SV inhibited protein kinase C (PKC) delta translocation induced by Ag-Ab but not by Tg or A23187. SV induced a reduction in active Rac1, and actin filament rearrangement. In conclusion, SV inhibits RBL-2H3 cell degranulation by inhibiting downstream signaling pathways, including the sequential degranulation pathway. These inhibitory effects were completely reversed by the addition of geranylgeraniol and might be induced by changes in the translocation of the small guanosine 5'-triphosphatase (GTPase) families Rab and Rho, which are related to vesicular transport PKC delta translocation and actin filament formation, respectively. These changes are caused by the inhibition of HMG-CoA reductase by SV following the synthesis of geranylgeranyl pyrophosphates, which play important roles in the activation of small GTPases, Rab.
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Degranulación de la Célula , Simvastatina , Animales , Ratas , Degranulación de la Célula/fisiología , Calcimicina/farmacología , Simvastatina/farmacología , Transducción de Señal , Mastocitos , Calcio/metabolismoRESUMEN
Evaluation of acrosome function in stallion sperm is mostly based on the use of inducers of acrosomal exocytosis (AE), such as the calcium ionophore A23187 or progesterone. Recently, it has been reported that incubation of stallion sperm under presumed capacitating conditions (i.e., medium formulated with calcium, bicarbonate, and bovine serum albumin) using a lactate-only containing medium (Lac-MW) results in a high rate of spontaneous AE in viable sperm (AE/Viable). In the current study, we developed an alternative assay of acrosome function for stallion sperm following the incubation of sperm in a medium formulated only with lactate as an energy substrate (Lac-MW). In Experiment 1, freshly ejaculated stallion sperm was incubated with 10 µM A23187, Lac-MW, or Control, for up to 6 h under capacitating conditions. The percentages of motile sperm, viable sperm, total AE (Total AE), and AE in viable sperm (AE/Viable) were compared among treatment groups. Incubation in Lac-MW, but not with Control or A23187, resulted in a time-dependent increase in the percentage of AE/Viable, as determined by flow cytometry, particularly at 4 and 6 h of incubation (P < 0.05). In Experiment 2, freshly ejaculated sperm was incubated in Lac-MW for up to 6 h, and the occurrence of protein tyrosine phosphorylation and AE/Viable were determined. At 4h and 6h of incubation in Lac-MW, â¼40% of the sperm displayed a protein tyrosine phosphorylation immunofluorescence pattern that coincides with that recently associated with stallion sperm capacitation (i.e., immunofluorescence signal at the acrosome and midpiece). In Experiment 3, the rate of AE/Viable sperm was compared among freshly ejaculated, cool-stored, and frozen/thawed stallion sperm. Except at 2h incubation in Lac-MW, differences in mean AE/Viable among fresh, cool-stored, and frozen/thawed sperm were not observed (P > 0.05). In Experiment 4, the relationship between Total AE (A23187), or AE/Viable (Lac-MW), and in vivo fertility of 5 stallions was determined. A linear relationship was observed between mean AE/Viable and the per-cycle (r = 0.93; P < 0.05) and seasonal (r = 0.66; P < 0.05) pregnancy rates of five stallions used for artificial insemination with cool-stored semen. In Experiment 5, frozen/thawed sperm from subfertile Thoroughbred (TB) stallions, known to carry the susceptibility genotype for Impaired Acrosomal Exocytosis (IAE; FKBP6 A/A-A/A) was evaluated following incubation in Lac-MW. Sperm from subfertile TB stallions with IAE had lower mean AE/Viable, at both 4h and 6h incubation in Lac-MW, when compared to that of fertile control stallions (P < 0.05). Overall, the Lac-MW model validated in the current study may be a useful complementary assay to evaluate the ability of stallion sperm to physiologically undergo AE and to study stallion fertility potential. This acrosome function assay can be used to evaluate fresh, cool-stored, or frozen/thawed stallion sperm, and describes a strong linear relationship with in vivo-fertility of stallions used in artificial insemination programs.
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Acrosoma , Semen , Embarazo , Femenino , Masculino , Caballos , Animales , Ácido Láctico , Calcimicina/farmacología , Espermatozoides/fisiología , Exocitosis , Tirosina , Motilidad EspermáticaRESUMEN
OBJECTIVE: To investigate whether empagliflozin could prevent injury-induced vascular neointimal hyperplasia and to further explore its mechanism. METHODS: Male C57BL/6J mice were divided into two groups with or without the empagliflozin treatment, and carotid ligation injury was performed to induce neointimal hyperplasia. The injured carotid arteries were collected for Western blotting (WB), histology and immunofluorescence analysis after four weeks. The inflammatory responses were analyzed by qRT-PCR to detect the inflammatory gene mRNA expression. To further explore its mechanism, HUVECs were treated with TGFß-1 to induce EndMT followed by empagliflozin or vehicle treatment in vitro. A23187 (Calcimycin), an agonist of NF-κB signaling was used in the experiment. RESULTS: The wall thickness and the neointima area was significantly reduced in the empagliflozin treatment group on day 28 after artery ligation. The Ki-67 positive cells were 28.33 ± 12.66% and 48.83 ± 10.41% in the empagliflozin-treated group and control group, respectively (P < 0.05). The mRNA expression levels of the inflammatory genes and inflammatory cells were decreased in the empagliflozin treatment group, as well as the MMP2 and MMP9. Meanwhile, empagliflozin can significantly reduce the migratory ability of inflammatory-treated HUVECs. The CD31 was increased in the TGFß1+empagliflozin group, whereas the FSP-1, phosphorylation of TAK-1 (p-TAK-1) and phosphorylation of NF-κB (p- NF-κB) expression level were decreased, compared to the control group without empagliflozin treatment. However, the expression level of FSP-1 and p-NF-κB were reversed after co-treatment with A23187, whereas the p-TAK-1 expression level was without any significant difference. CONCLUSION: Empagliflozin inhibits the inflammation-induced EndMT via the TAK-1/NF-κB signaling pathway.
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FN-kappa B , Lesiones del Sistema Vascular , Ratones , Animales , Masculino , FN-kappa B/metabolismo , Hiperplasia , Neointima/tratamiento farmacológico , Calcimicina , Ratones Endogámicos C57BL , ARN MensajeroRESUMEN
PURPOSE: Despite the success of ICSI in treating severe male factor infertile patients, total fertilization failure (FF) still occurs in around 1-3% of ICSI cycles. To overcome FF, the use of calcium ionophores has been proposed to induce oocyte activation and restore fertilization rates. However, assisted oocyte activation (AOA) protocols and ionophores vary between laboratories, and the morphokinetic development underlying AOA remains understudied. METHODS: A prospective single-center cohort study involving 81 in vitro matured metaphase-II oocytes from 66 oocyte donation cycles artificially activated by A23187 (GM508 CultActive, Gynemed) (n=42) or ionomycin (n=39). Parthenogenesis was induced, and morphokinetic parameters (tPNa, tPNf, t2-t8, tSB, and tB) were compared between the 2 study groups and a control group comprising 39 2PN-zygotes from standard ICSI cycles. RESULTS: Ionomycin treatment resulted in higher activation rates compared to A23187 (38.5% vs 23.8%, p=0.15). Importantly, none of the A23187-activated parthenotes formed blastocysts. When evaluating the morphokinetic dynamics between the two ionophores, we found that tPNa and tPNf were significantly delayed in the group treated by A23187 (11.84 vs 5.31, p=0.002 and 50.15 vs 29.69, p=0.005, respectively). t2 was significantly delayed in A23187-activated parthenotes when compared to the double heterologous control embryo group. In contrast, the morphokinetic development of ionomycin-activated parthenotes was comparable to control embryos (p>0.05). CONCLUSION: Our results suggest that A23187 leads to lower oocyte activation rates and profoundly affects morphokinetic timings and preimplantation development in parthenotes. Despite our limited sample size and low parthenote competence, standardization and further optimization of AOA protocols may allow wider use and improved outcomes for FF cycles.
Asunto(s)
Oocitos , Inyecciones de Esperma Intracitoplasmáticas , Masculino , Animales , Ionomicina/farmacología , Ionóforos/farmacología , Calcimicina/farmacología , Estudios de Cohortes , Inyecciones de Esperma Intracitoplasmáticas/métodosRESUMEN
Cryopreservation significantly alters the phenotype of platelets; generating distinct subpopulations, which may influence the formation of platelet leukocyte aggregates (PLA). PLAs are immunomodulatory and have been associated with transfusion-associated adverse events. As such, the aim of this study was to examine the effect of cryopreservation on the ability of platelets to form PLAs, using a monocyte-like cell line (THP-1). Platelets were tested pre-freeze, post-thaw and following stimulation with TRAP-6 or A23187, both alone and following co-culture with THP-1 cells for 1 and 24 hours (n = 6). Platelet subpopulations and platelet-THP-1 cell aggregates were analyzed using multi-color imaging flow cytometry using Apotracker Green (ApoT), CD42b, CD62P, CD61, and CD45. Cryopreservation resulted in the generation of activated (ApoT-/CD42b+/CD62P+), procoagulant (ApoT+/CD42b+/CD62P+) and a novel (ApoT+/CD42b+/CD62P-) platelet subpopulation. Co-incubation of cryopreserved platelets with THP-1 cells increased PLA formation compared to pre-freeze but not TRAP-6 or A23187 stimulated platelets. P-selectin on the surface membrane was correlated with increased PLA formation. Our findings demonstrate that cryopreservation increases the interaction between platelets and THP-1 cells, largely due to an increase in procoagulant platelets. Further investigation is required to determine the immunological consequences of this interaction.
What do we know? Cryopreserved platelets are an alternative to overcome issues with the short shelf-life of room-temperature stored plateletsAfter thawing, cryopreserved platelets exhibit changes in cell structure and receptor abundanceActivated platelets can attach to leukocytes, forming platelet-leukocyte aggregates and altering their immune functionPlatelet-leukocyte aggregates can increase inflammation, which is associated with adverse events after transfusion, which can negatively affect patient outcomesWhat did we discover? Cryopreservation results in a heterogenous mix of platelet subpopulationsCryopreserved platelets display increased adherence to a monocyte-like cell line (THP-1 cells). Platelet-THP-1 aggregate formation was linked to expression of CD62P on the surface of the plateletsThe increase in cryopreserved platelet-THP-1 cell aggregates was largely due to an increase in procoagulant plateletsWhat is the impact? Our data demonstrate that cryopreservation increases platelet interaction with a monocyte-like cell lineThis may mediate immune responses and/or circulation time of transfused platelets.
Asunto(s)
Plaquetas , Monocitos , Calcimicina/metabolismo , Calcimicina/farmacología , Plaquetas/metabolismo , Monocitos/metabolismo , Fenotipo , Criopreservación/métodos , Poliésteres/metabolismo , Selectina-P/metabolismo , Activación PlaquetariaRESUMEN
STUDY QUESTION: Can recurrent embryo developmental problems after ICSI be overcome by assisted oocyte activation (AOA)? SUMMARY ANSWER: AOA did not improve blastocyst formation in our patient cohort with recurrent embryo developmental problems after ICSI. WHAT IS KNOWN ALREADY: The use of AOA to artificially induce calcium (Ca2+) rises by using Ca2+ ionophores (mainly calcimycin and ionomycin) has been reported as very effective in overcoming fertilization failure after ICSI, especially in patients whose Ca2+ dynamics during fertilization are deficient. However, there is only scarce and contradictory literature on the use of AOA to overcome embryo developmental problems after ICSI, and it is not clear whether abnormal Ca2+ patterns during fertilization disturb human preimplantation embryo development. Moreover, poor embryo development after ICSI has also been linked to genetic defects in the subcortical maternal complex (SCMC) genes. STUDY DESIGN, SIZE, DURATION: This prospective cohort single-center study compared ICSI-AOA cycles and previous ICSI cycles in couples with normal fertilization rates (≥60%) but impaired embryonic development (≤15% blastocyst formation) in at least two previous ICSI cycles. In total, 42 couples with embryo developmental problems were included in this study from January 2018 to January 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS: Of the 42 couples included, 17 underwent an ICSI-AOA cycle consisting of CaCl2 injection and double ionomycin exposure. Fertilization, blastocyst development, pregnancy, and live birth rates after ICSI-AOA were compared to previous ICSI cycles. In addition, the calcium pattern induced by the male patient's sperm was investigated by mouse oocyte calcium analysis. Furthermore, all 42 couples underwent genetic screening. Female patients were screened for SCMC genes (TLE6, PADI6, NLRP2, NLRP5, NLRP7, and KHDC3L) and male patients were screened for the sperm-oocyte-activating factor PLCZ1. MAIN RESULTS AND THE ROLE OF CHANCE: We compared 17 AOA cycles to 44 previous ICSI cycles from the same patient cohort. After AOA, a total fertilization rate of 68.95% (131/190), a blastocyst development rate of 13.74% (18/131), a pregnancy rate of 29.41% (5/17), and a live birth rate of 23.53% (4/17) were achieved, which was not different from the previous ICSI cycles (76.25% (321/421, P-value = 0.06); 9.35% (30/321, P-value = 0.18), 25.00% (11/44, P-value = 0.75), and 15.91% (7/44, P-value = 0.48), respectively). Calcium analysis showed that patient's sperm induced calcium patterns similar to control sperm samples displaying normal embryo developmental potential. Genetic screening revealed 10 unique heterozygous variants (in NLRP2, NLRP5, NLRP7, TLE6, and PADI6) of uncertain significance (VUS) in 14 females. Variant NLRP5 c.623-12_623-11insTTC (p.?) was identified in two unrelated individuals and variant NLRP2 c.1572T>C (p.Asp524=) was identified in four females. Interestingly, we identified a previously reported homozygous mutation PLCZ1, c.1499C>T (p.Ser500Leu), in a male patient displaying impaired embryonic development, but not showing typical fertilization failure. LIMITATIONS, REASONS FOR CAUTION: Our strict inclusion criteria, requiring at least two ICSI cycles with impaired embryo development, reduced cycle-to-cycle variability, while the requirement of a lower blastocyst development not influenced by a poor fertilization excluded couples who otherwise would be selective cases for AOA; however, these criteria limited the sample size of this study. Targeted genetic screening might be too restricted to identify a genetic cause underlying the phenotype of poor embryo development for all patients. Moreover, causality of the identified VUS should be further determined. WIDER IMPLICATIONS OF THE FINDINGS: Strong evidence for AOA overcoming impaired embryonic development is still lacking in the literature. Thus far, only one article has reported a beneficial effect of AOA (using calcimycin) compared to previous ICSI cycles in this patient population, whilst two more recent sibling-oocyte control studies (one using calcimycin and the other ionomycin) and our research (using ionomycin) could not corroborate these findings. Although no major abnormalities have been found in children born after AOA, this technique should be reserved for couples with a clear Ca2+-release deficiency. Finally, genetic screening by whole-exome sequencing may reveal novel genes and variants linked to embryo developmental problems and allow the design of more personalized treatment options, such as wild-type complementary RNA or recombinant protein injection. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the Flemish Fund for Scientific Research (grant FWO.OPR.2015.0032.01 to B.H. and grant no. 1298722N to A.B.). A.C.B., D.B., A.B., V.T., R.P., F.M., I.D.C., L.L., D.S., P.D.S., P.C., and F.V.M. have nothing to disclose. B.H. reports a research grant from the Flemish Fund for Scientific Research and reports being a board member of the Belgian Society for Reproductive Medicine and the Belgian Ethical Committee on embryo research. TRIAL REGISTRATION NUMBER: NCT03354013.
Asunto(s)
Calcio , Inyecciones de Esperma Intracitoplasmáticas , Embarazo , Niño , Humanos , Masculino , Femenino , Animales , Ratones , Inyecciones de Esperma Intracitoplasmáticas/métodos , Ionomicina , Calcimicina , Estudios Prospectivos , Semen , Índice de Embarazo , Oocitos , Desarrollo Embrionario , Estudios Retrospectivos , Proteínas Adaptadoras Transductoras de Señales , Proteínas Reguladoras de la ApoptosisRESUMEN
BACKGROUND: Platelets play a modulatory role in inflammatory response by secreting a vast array of granules and disintegrating into membrane-bound microparticles upon activation. The interplay between eosinophils and platelets is postulated to be implicated in the pathology of allergic airway inflammation. In this study, we investigated whether activated platelets can induce eosinophil extracellular trap (EET) formation, a cellular process by which activated eosinophils release net-like DNA fibers. METHODS: Platelets were stimulated with the calcium ionophore, A23187, and the platelet agonists, thrombin and adenosine diphosphate (ADP). Platelet cultures were fractionated into conditioned medium (CM) and pellet, which were then overlaid on eosinophils to examine EET formation. RESULTS: The CM and pellet from A23187-activated platelets stimulated eosinophils to generate EET, whereas those from thrombin- or ADP-activated platelets failed to induce such generation. The EET-inducing activity of the A23187-activated platelet culture was linearly proportional to the number of activated platelets. Interestingly, while EET formation induced by the direct stimulation of eosinophils with A23187 was NADPH oxidase (NOX)-dependent, EET formation induced by A23187-activated platelets was NOX-independent and significantly inhibited by necroptosis pathway inhibitors. CONCLUSIONS: Activated platelets and their products may induce EET formation, thereby potentiating their role in eosinophilic airway inflammation.