RESUMEN
The gangliosidoses GM2 are a group of pathologies mainly affecting the central nervous system due to the impaired GM2 ganglioside degradation inside the lysosome. Under physiological conditions, GM2 ganglioside is catabolized by the ß-hexosaminidase A in a GM2 activator protein-dependent mechanism. In contrast, uncharged substrates such as globosides and some glycosaminoglycans can be hydrolyzed by the ß-hexosaminidase B. Monogenic mutations on HEXA, HEXB, or GM2A genes arise in the Tay-Sachs (TSD), Sandhoff (SD), and AB variant diseases, respectively. In this work, we validated a CRISPR/Cas9-based gene editing strategy that relies on a Cas9 nickase (nCas9) as a potential approach for treating GM2 gangliosidoses using in vitro models for TSD and SD. The nCas9 contains a mutation in the catalytic RuvC domain but maintains the active HNH domain, which reduces potential off-target effects. Liposomes (LPs)- and novel magnetoliposomes (MLPs)-based vectors were used to deliver the CRISPR/nCas9 system. When LPs were used as a vector, positive outcomes were observed for the ß-hexosaminidase activity, glycosaminoglycans levels, lysosome mass, and oxidative stress. In the case of MLPs, a high cytocompatibility and transfection ratio was observed, with a slight increase in the ß-hexosaminidase activity and significant oxidative stress recovery in both TSD and SD cells. These results show the remarkable potential of CRISPR/nCas9 as a new alternative for treating GM2 gangliosidoses, as well as the superior performance of non-viral vectors in enhancing the potency of this therapeutic approach.
Asunto(s)
Gangliosidosis GM2 , Enfermedad de Tay-Sachs , Desoxirribonucleasa I/metabolismo , Fibroblastos/metabolismo , Proteína Activadora de G (M2) , Gangliósido G(M2)/genética , Gangliósido G(M2)/metabolismo , Gangliosidosis GM2/genética , Gangliosidosis GM2/metabolismo , Gangliosidosis GM2/terapia , Edición Génica , Globósidos/metabolismo , Glicosaminoglicanos/metabolismo , Hexosaminidasa A/metabolismo , Humanos , Lipopolisacáridos/metabolismo , Liposomas/metabolismo , Enfermedad de Tay-Sachs/genética , Enfermedad de Tay-Sachs/metabolismo , Enfermedad de Tay-Sachs/terapia , beta-N-Acetilhexosaminidasas/metabolismoRESUMEN
AIM: We determined the role played by O-linked N-acetylglucosamine (O-GlcNAc) of proteins in systemic arteries during late pregnancy in normotensive and hypertensive rats. MAIN METHODS: O-GlcNAc levels and O-GlcNAc modification of endothelial nitric oxide synthase (eNOS) were determined in aorta (conductance vessel) and mesenteric arteries (resistance vessels) of non-pregnant (NP) and pregnant (P) Wistar rats and spontaneously hypertensive rats (SHR). Vascular O-GlcNAc-modified proteins, O-GlcNAcase (OGA) and O-GlcNAc transferase (OGT) expression, and OGA activity were analyzed. Concentration-response to phenylephrine (PE) curves were constructed for arteries with and without endothelium. Arteries were treated with vehicle or PugNAc (OGA inhibitor, 100 µmol/L) in the presence of L-NAME (NOS inhibitor, 100 µmol/L). KEY FINDINGS: The content of vascular O-GlcNAc-modified proteins was lower, OGT and OGA expression did not change, and OGA activity was higher in arteries of P-Wistar rats and P-SHR compared to arteries of NP-groups. Reactivity to PE increased in arteries of P-Wistar rats treated with PugNAc compared to vehicle. O-GlcNAcylation of eNOS decreased in P-SHR compared to NP-SHR. PugNAc partially inhibited the effects of endothelium removal and L-NAME on reactivity to PE in arteries of P-Wistar rats. However, PugNAc did not alter reactivity to PE in arteries of P-SHR. Our data showed that pregnancy decreased the content of vascular O-GlcNAc-modified proteins. SIGNIFICANCE: Increased OGA activity and decreased O-GlcNAc modification of eNOS boosts eNOS activity in arteries of P-Wistar rats. In P-SHR, altered OGA activity may lower the content of O-GlcNAc-modified proteins, but decreased OGT activity seems a potential mechanism to reduce glycosylation.
Asunto(s)
Acetilglucosamina/química , Aorta Torácica/fisiopatología , Hipertensión/fisiopatología , Arterias Mesentéricas/fisiopatología , Procesamiento Proteico-Postraduccional , beta-N-Acetilhexosaminidasas/metabolismo , Animales , Aorta Torácica/enzimología , Femenino , Glicosilación , Hipertensión/enzimología , Arterias Mesentéricas/enzimología , N-Acetilglucosaminiltransferasas , Embarazo , Ratas , Ratas Endogámicas SHR , Ratas Wistar , beta-N-Acetilhexosaminidasas/químicaRESUMEN
The main objectives of this study were to evaluate the chemical constitution and allergenic potential of red propolis extract (RPE). They were evaluated, using high performance liquid chromatography (HPLC) and the release of ß-hexosaminidase, respectively. A plethora of biologically active polyphenols and the absence of allergic responses were evinced. RPE inhibited the release of ß-hexosaminidase, suggesting that the extract does not stimulate allergic responses. Additionally, the physicochemical properties and antibacterial activity of hydrogel membranes loaded with RPE were analyzed. Bio-polymeric hydrogel membranes (M) were obtained using 5% carboxymethylcellulose (M1 and M2), 1.0% of citric acid (M3) and 10% RPE (for all). Their characterization was performed using thermal analysis, Fourier transform infrared (FTIR), total phenolic content, phenol release test and, antioxidant activity through 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) and Ferric Reducing Antioxidant Power (FRAP). The latter appointed to the similar antioxidant capacity of the M1, M2 and M3. The degradation profiles showed higher thermostability to M3, followed by M2 and M1. The incorporation of RPE into the matrices and the crosslinking of M3 were evinced by FTIR. There were differences in the release of phenolic compounds, with a higher release related to M1 and lower in the strongly crosslinked M3. The degradation profiles showed higher thermostability to M3, followed by M2 and M1. The antibacterial activity of the membranes was determined using the disc diffusion assay, in comparison with controls, obtained in the same way, without RPE. The membranes elicited antibacterial activity against Staphylococcus aureus and Staphylococcus epidermidis, with superior performance over M3. The hydrogel membranes loaded with RPE promote a physical barrier against bacterial skin infections and may be applied in the wound healing process.
Asunto(s)
Própolis/química , Administración Tópica , Alérgenos/química , Animales , Antibacterianos/administración & dosificación , Antibacterianos/química , Antibacterianos/farmacología , Antioxidantes/administración & dosificación , Antioxidantes/química , Antioxidantes/farmacología , Vendajes , Materiales Biocompatibles/administración & dosificación , Materiales Biocompatibles/química , Materiales Biocompatibles/farmacología , Biopolímeros/administración & dosificación , Biopolímeros/química , Biopolímeros/farmacología , Brasil , Línea Celular , Fenómenos Químicos , Cromatografía Líquida de Alta Presión , Composición de Medicamentos , Estabilidad de Medicamentos , Humanos , Hidrogeles , Técnicas In Vitro , Mastocitos/efectos de los fármacos , Mastocitos/enzimología , Mastocitos/inmunología , Membranas Artificiales , Fenoles/química , Própolis/administración & dosificación , Própolis/farmacología , Ratas , Espectroscopía Infrarroja por Transformada de Fourier , Staphylococcus aureus/efectos de los fármacos , Staphylococcus epidermidis/efectos de los fármacos , Termogravimetría , beta-N-Acetilhexosaminidasas/metabolismoRESUMEN
Zika virus (ZIKV) is an emergent arthropod-borne virus whose outbreak in Brazil has brought major public health problems. Infected individuals have different symptoms, including rash and pruritus, which can be relieved by the administration of antiallergics. In the case of pregnant women, ZIKV can cross the placenta and infect the fetus leading to congenital defects. We have identified that mast cells in the placentae of patients who had Zika during pregnancy can be infected. This led to our investigation on the possible role of mast cells during a ZIKV infection, using the HMC-1 cell line. We analyzed their permissiveness to infection, release of mediators and ultrastructural changes. Flow cytometry detection of ZIKV-NS1 expression 24 h post infection in 45.3% of cells showed that HMC-1 cells are permissive to ZIKV infection. Following infection, ß-hexosaminidase was measured in the supernatant of the cells with a notable release at 30 min. In addition, an increase in TNF-α, IL-6, IL-10 and VEGF levels were measured at 6 h and 24 h post infection. Lastly, different intracellular changes were observed in an ultrastructural analysis of infected cells. Our findings suggest that mast cells may represent an important source of mediators that can activate other immune cell types during a ZIKV infection, which has the potential to be a major contributor in the spread of the virus in cases of vertical transmission.
Asunto(s)
Citocinas/metabolismo , Mastocitos/inmunología , Infección por el Virus Zika/inmunología , Virus Zika/inmunología , Adulto , Brasil , Línea Celular , Femenino , Humanos , Inmunohistoquímica , Transmisión Vertical de Enfermedad Infecciosa , Interleucina-10/metabolismo , Interleucina-6/metabolismo , Mastocitos/patología , Mastocitos/ultraestructura , Mastocitos/virología , Microscopía Electrónica de Transmisión , Placenta/inmunología , Placenta/metabolismo , Placenta/virología , Embarazo , Factor de Necrosis Tumoral alfa/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Virus Zika/patogenicidad , Infección por el Virus Zika/enzimología , Infección por el Virus Zika/fisiopatología , Infección por el Virus Zika/transmisión , beta-N-Acetilhexosaminidasas/metabolismoRESUMEN
Macrophages contribute to a continuous increase in blood pressure and kidney damage in hypertension, but their polarization status and the underlying mechanisms have not been clarified. This study revealed an important role for M2 macrophages and the YM1/Chi3l3 protein in hypertensive nephropathy in a mouse model of hypertension. Bone marrow cells were isolated from the femurs and tibia of male FVB/N (control) and transgenic hypertensive animals that overexpressed the rat form of angiotensinogen (TGM(rAOGEN)123, TGM123-FVB/N). The cells were treated with murine M-CSF and subsequently with LPS+IFN-γ to promote their polarization into M1 macrophages and IL-4+IL-13 to trigger the M2 phenotype. We examined the kidneys of TGM123-FVB/N animals to assess macrophage polarization and end-organ damage. mRNA expression was evaluated using real-time PCR, and protein levels were assessed through ELISA, CBA, Western blot, and immunofluorescence. Histology confirmed high levels of renal collagen. Cells stimulated with LPS+IFN-γ in vitro showed no significant difference in the expression of CD86, an M1 marker, compared to cells from the controls or the hypertensive mice. When stimulated with IL-4+IL-13, however, macrophages of the hypertensive group showed a significant increase in CD206 expression, an M2 marker. The M2/M1 ratio reached 288%. Our results indicate that when stimulated in vitro, macrophages from hypertensive mice are predisposed toward polarization to an M2 phenotype. These data support results from the kidneys where we found an increased infiltration of macrophages predominantly polarized to M2 associated with high levels of YM1/Chi3l3 (91,89%), suggesting that YM1/Chi3l3 may be a biomarker of hypertensive nephropathy.
Asunto(s)
Hipertensión/metabolismo , Enfermedades Renales/metabolismo , Lectinas/metabolismo , Macrófagos/metabolismo , beta-N-Acetilhexosaminidasas/metabolismo , Animales , Biomarcadores/metabolismo , Interleucina-13/metabolismo , Interleucina-4/metabolismo , Riñón/metabolismo , Enfermedades Renales/genética , Lectinas/genética , Activación de Macrófagos/fisiología , Masculino , Ratones , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , beta-N-Acetilhexosaminidasas/genéticaRESUMEN
Mast cells play an essential role in different immunological phenomena including allergy and infectious diseases. Several bacteria induce mast cell activation leading to degranulation and the production of several cytokines and chemokines. However, mast cells also have different microbicidal activities such as phagocytosis and the release of DNA with embedded granular proteins known as Mast Cell Extracellular Traps (MCETs). Although previous reports indicate that extracellular bacteria are able to induce MCETs little is known if intracellular bacteria can induce these structures. In this work, we evaluated MCETs induction by the intracellular bacteria Listeria monocytogenes. We found that mast cells released DNA after stimulation with L. monocytogenes, and this DNA was complexed to histone and tryptase. Before extracellular DNA release, L. monocytogenes induced modifications to the mast cell nuclear envelope and DNA was detected outside the nucleus. L. monocytogenes stimulated mast cells to produce significant amounts of reactive oxygen species (ROS) and blocking NADPH oxidase diminished DNA release by mast cells. Finally, MCETs showed antimicrobial activity against L. monocytogenes that was partially blocked when ß-hexosaminidase activity was inhibited. These results show that L. monocytogenes induces mast cells to produce microbicidal MCETs, suggesting a role for mast cells in containing infection beyond the induction of inflammation.
Asunto(s)
Trampas Extracelulares/inmunología , Trampas Extracelulares/metabolismo , Interacciones Huésped-Patógeno/inmunología , Listeria monocytogenes/inmunología , Mastocitos/inmunología , Mastocitos/metabolismo , Línea Celular , ADN/metabolismo , Histonas/metabolismo , Humanos , Listeriosis , Mastocitos/ultraestructura , Membrana Nuclear/ultraestructura , Fagocitosis/inmunología , Especies Reactivas de Oxígeno/metabolismo , beta-N-Acetilhexosaminidasas/metabolismoRESUMEN
O-GlcNAcylation is a dynamic post-translational modification consisting of the addition of a single N-acetylglucosamine sugar to serine and threonine residues in proteins by the enzyme O-linked ß-N-acetylglucosamine transferase (OGT), whereas the enzyme O-GlcNAcase (OGA) removes the modification. In cancer, tumor samples present with altered O-GlcNAcylation; however, changes in O-GlcNAcylation are not consistent between tumor types. Interestingly, the tumor suppressor p53 is modified by O-GlcNAc, and most solid tumors contain mutations in p53 leading to the loss of p53 function. Because ovarian cancer has a high frequency of p53 mutation rates, we decided to investigate the relationship between O-GlcNAcylation and p53 function in ovarian cancer. We measured a significant decrease in O-GlcNAcylation of tumor tissue in an ovarian tumor microarray. Furthermore, O-GlcNAcylation was increased, and OGA protein and mRNA levels were decreased in ovarian tumor cell lines not expressing the protein p53. Treatment with the OGA inhibitor Thiamet-G (TMG), silencing of OGA, or overexpression of OGA and OGT led to p53 stabilization, increased nuclear localization, and increased protein and mRNA levels of p53 target genes. These data suggest that changes in O-GlcNAc homeostasis activate the p53 pathway. Combination treatment of the chemotherapeutic cisplatin with TMG decreased tumor cell growth and enhanced cell cycle arrest without impairing cytotoxicity. The effects of TMG on tumor cell growth were partially dependent on wild type p53 activation. In conclusion, changes in O-GlcNAc homeostasis activate the wild type p53 pathway in ovarian cancer cells, and OGA inhibition has the potential as an adjuvant treatment for ovarian carcinoma.
Asunto(s)
Acetilglucosamina/metabolismo , Núcleo Celular/metabolismo , Homeostasis , Neoplasias Ováricas/metabolismo , Procesamiento Proteico-Postraduccional , Proteína p53 Supresora de Tumor/metabolismo , Acetilglucosamina/genética , Transporte Activo de Núcleo Celular/efectos de los fármacos , Transporte Activo de Núcleo Celular/genética , Línea Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/patología , Femenino , Silenciador del Gen , Humanos , Mutación , N-Acetilglucosaminiltransferasas/genética , N-Acetilglucosaminiltransferasas/metabolismo , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Estabilidad Proteica/efectos de los fármacos , Piranos/farmacología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Neoplásico/biosíntesis , ARN Neoplásico/genética , Tiazoles/farmacología , Proteína p53 Supresora de Tumor/genética , beta-N-Acetilhexosaminidasas/antagonistas & inhibidores , beta-N-Acetilhexosaminidasas/genética , beta-N-Acetilhexosaminidasas/metabolismoRESUMEN
BACKGROUND: Mast cells are hematopoietically derived cells that play a role in inflammatory processes such as allergy, as well as in the immune response against pathogens by the selective and rapid release of preformed and lipid mediators, and the delayed release of cytokines. The native homotetrameric lectin ArtinM, a D-mannose binding lectin purified from Artocarpus heterophyllus seeds, is one of several lectins that are able to activate mast cells. Besides activating mast cells, ArtinM has been shown to affect several biological responses, including immunomodulation and acceleration of wound healing. Because of the potential pharmacological application of ArtinM, a recombinant ArtinM (rArtinM) was produced in Escherichia coli. The current study evaluated the ability of rArtinM to induce mast cell degranulation and activation. RESULTS: The glycan binding specificity of rArtinM was similar to that of jArtinM. rArtinM, via its CRD, was able to degranulate, releasing ß-hexosaminidase and TNF-α, and to promote morphological changes on the mast cell surface. Moreover, rArtinM induced the release of the newly-synthesized mediator, IL-4. rArtinM does not have a co-stimulatory effect on the FcεRI degranulation via. The IgE-dependent mast cell activation triggered by rArtinM seems to be dependent on NFkB activation. CONCLUSIONS: The lectin rArtinM has the ability to activate and degranulate mast cells via their CRDs. The present study indicates that rArtinM is a suitable substitute for the native form, jArtinM, and that rArtinM may serve as an important and reliable pharmacological agent.
Asunto(s)
Mastocitos/inmunología , Lectinas de Plantas/inmunología , Proteínas Recombinantes/inmunología , Animales , Artocarpus/inmunología , Degranulación de la Célula , Línea Celular , Clonación Molecular , Escherichia coli/genética , Histamina/metabolismo , Inmunoglobulina E/metabolismo , Inmunomodulación , Interleucina-4/metabolismo , Manosa/metabolismo , FN-kappa B/metabolismo , Lectinas de Plantas/aislamiento & purificación , Unión Proteica , Ratas , Proteínas Recombinantes/aislamiento & purificación , beta-N-Acetilhexosaminidasas/metabolismoRESUMEN
Obesity and high fat intake induce alterations in vascular function and structure. Aberrant O-GlcNAcylation (O-GlcNAc) of vascular proteins has been implicated in vascular dysfunction associated with cardiovascular and metabolic diseases. In the present study, we tested the hypothesis that high-fat diet (HFD)-mediated increases in O-GlcNAc-modified proteins contribute to cerebrovascular dysfunction. O-GlcNAc-protein content was increased in arteries from male Wistar rats treated with a HFD (45% fat) for 12 weeks compared with arteries from rats on control diet (CD). HFD augmented body weight [(g) 550±10 compared with 502±10 CD], increased plasma triacylglycerols [(mg/dl) 160±20 compared with 95±15 CD] and increased contractile responses of basilar arteries to serotonin [5-hydroxytryptamine (5-HT)] [(pD2) 7.0±0.1 compared with 6.7±0.09 CD] and the thromboxane analogue 9,11-dideoxy-9α,11α-methanoepoxy prostaglandin F2α (U-46619) [(pD2) 7.2±0.1 compared with 6.8±0.09 CD]. Of importance, increased levels of O-GlcNAc [induced by 24 h-incubation of vessels with a potent inhibitor of O-GlcNAcase (OGA), O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PugNAc)] increased basilar artery contractions in response to U-46619 [(pD2) 7.4±0.07 compared with 6.8±0.08 CD] and 5-HT [(pD2) 7.5±0.06 compared with 7.1±0.1 CD]. Vessels from rats on the HFD for 12 weeks and vessels treated with PugNAc displayed increased phosphorylation of p38 (Thr(180/182)) and extracellular signal-regulated kinase 1/2 (Erk1/2) (Ser(180/221)). Increased 5HT-induced contractions in arteries from rats on the HFD or in arteries incubated with PugNAc were abrogated by mitogen-activated protein kinase (MAPK) inhibitors. Our data show that HFD augments cerebrovascular O-GlcNAc and this modification contributes to increased contractile responses and to the activation of the MAPK pathway in the rat basilar artery.
Asunto(s)
Acetilglucosamina/metabolismo , Arterias Cerebrales/metabolismo , Dieta Alta en Grasa , Hiperlipidemias/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Obesidad/metabolismo , beta-N-Acetilhexosaminidasas/metabolismo , Animales , Masculino , N-Acetilglucosaminiltransferasas/metabolismo , Fosforilación/fisiología , Procesamiento Proteico-Postraduccional/fisiología , Ratas WistarRESUMEN
Mucopolysaccharidosis (MPS) is a group of lysosomal storage diseases (LSD), characterized by the deficiency of a lysosomal enzyme responsible for the degradation of glycosaminoglycans (GAG). This deficiency leads to the lysosomal accumulation of partially degraded GAG. Nevertheless, deficiency of a single lysosomal enzyme has been associated with impairment in other cell mechanism, such as apoptosis and redox balance. Although GAG analysis represents the main biomarker for MPS diagnosis, it has several limitations that can lead to a misdiagnosis, whereby the identification of new biomarkers represents an important issue for MPS. In this study, we used a system biology approach, through the use of a genome-scale human metabolic reconstruction to understand the effect of metabolism alterations in cell homeostasis and to identify potential new biomarkers in MPS. In-silico MPS models were generated by silencing of MPS-related enzymes, and were analyzed through a flux balance and variability analysis. We found that MPS models used approximately 2286 reactions to satisfy the objective function. Impaired reactions were mainly involved in cellular respiration, mitochondrial process, amino acid and lipid metabolism, and ion exchange. Metabolic changes were similar for MPS I and II, and MPS III A to C; while the remaining MPS showed unique metabolic profiles. Eight and thirteen potential high-confidence biomarkers were identified for MPS IVB and VII, respectively, which were associated with the secondary pathologic process of LSD. In vivo evaluation of predicted intermediate confidence biomarkers (ß-hexosaminidase and ß-glucoronidase) for MPS IVA and VI correlated with the in-silico prediction. These results show the potential of a computational human metabolic reconstruction to understand the molecular mechanisms this group of diseases, which can be used to identify new biomarkers for MPS.
Asunto(s)
Mucopolisacaridosis/metabolismo , Biomarcadores/metabolismo , Simulación por Computador , Células HEK293 , Humanos , Leucocitos Mononucleares/enzimología , Análisis de Flujos Metabólicos , Redes y Vías Metabólicas , Biología de Sistemas , beta-N-Acetilhexosaminidasas/metabolismoRESUMEN
Trypanosoma cruzi, the etiological agent of Chagas' disease, induces a persistent inflammatory response. Macrophages are a first line cell phenotype involved in the clearance of infection. Upon parasite uptake, these cells increase inflammatory mediators like NO, TNF-α, IL-1ß and IL-6, leading to parasite killing. Although desired, inflammatory response perpetuation and exacerbation may lead to tissue damage. Peroxisome proliferator-activated receptors (PPARs) are ligand-dependent nuclear transcription factors that, besides regulating lipid and carbohydrate metabolism, have a significant anti-inflammatory effect. This is mediated through the interaction of the receptors with their ligands. PPARγ, one of the PPAR isoforms, has been implicated in macrophage polarization from M1, the classically activated phenotype, to M2, the alternatively activated phenotype, in different models of metabolic disorders and infection. In this study, we show for the first time that, besides PPARγ, PPARα is also involved in the in vitro polarization of macrophages isolated from T. cruzi-infected mice. Polarization was evidenced by a decrease in the expression of NOS2 and proinflammatory cytokines and the increase in M2 markers like Arginase I, Ym1, mannose receptor and TGF-ß. Besides, macrophage phagocytic activity was significantly enhanced, leading to increased parasite load. We suggest that modulation of the inflammatory response by both PPARs might be due, at least in part, to a change in the profile of inflammatory macrophages. The potential use of PPAR agonists as modulators of overt inflammatory response during the course of Chagas' disease deserves further investigation.
Asunto(s)
Enfermedad de Chagas/metabolismo , Macrófagos/metabolismo , PPAR alfa/metabolismo , PPAR gamma/metabolismo , Animales , Arginasa/genética , Arginasa/metabolismo , Western Blotting , Células Cultivadas , Enfermedad de Chagas/genética , Enfermedad de Chagas/parasitología , Citocinas/genética , Citocinas/metabolismo , Interacciones Huésped-Patógeno , Mediadores de Inflamación/metabolismo , Lectinas/genética , Lectinas/metabolismo , Ligandos , Activación de Macrófagos/efectos de los fármacos , Macrófagos/clasificación , Macrófagos/efectos de los fármacos , Masculino , Ratones Endogámicos BALB C , Microscopía Fluorescente , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , PPAR alfa/genética , PPAR gamma/genética , Fagocitosis/efectos de los fármacos , Prostaglandina D2/análogos & derivados , Prostaglandina D2/farmacología , Pirimidinas/farmacología , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Trypanosoma cruzi/fisiología , beta-N-Acetilhexosaminidasas/genética , beta-N-Acetilhexosaminidasas/metabolismoRESUMEN
The aim of this study was to determine whether hydroxytyrosol and oleuropein, the major phenols found in olives and olive oil, inhibit mast cell activation induced by immune and non-immune pathways. Purified peritoneal mast cells were preincubated in the presence of test compounds (hydroxytyrosol or oleuropein), before incubation with concanavalin A, compound 48/80 or calcium ionophore A23187. Dose-response and time-dependence studies were carried out. Comparative studies with sodium cromoglycate, a classical mast cell stabilizer, were also made. After incubation the supernatants and pellets were used to determine the ß-hexosaminidase content by colorimetric reaction. The percentage of ß-hexosaminidase release in each tube was calculated and taken as a measure of mast cell activation. Other samples of cell pellets were used for cell viability studies by the trypan blue dye exclusion test, or fixed for light and electron microscopy. Biochemical and morphological findings of the present study showed for the first time that hydroxytyrosol and oleuropein inhibit mast cell degranulation induced by both immune and non-immune pathways. These results suggest that olive phenols, particularly hydroxytyrosol and oleuropein, may provide insights into the development of useful tools for the prevention and treatment of mast cell-mediated disorders.
Asunto(s)
Degranulación de la Célula/efectos de los fármacos , Iridoides/farmacología , Mastocitos/efectos de los fármacos , Alcohol Feniletílico/análogos & derivados , Animales , Relación Dosis-Respuesta a Droga , Glucósidos Iridoides , Masculino , Aceite de Oliva , Alcohol Feniletílico/farmacología , Aceites de Plantas/química , Ratas Wistar , beta-N-Acetilhexosaminidasas/metabolismoRESUMEN
The GlcNAcstatin is a potent inhibitor of O-glycoprotein 2-acetamino-2-deoxy-ß-D-glucopyranosidase, which has been related with type II diabetes and neurodegenerative disorders. Herein, hybrid quantum mechanics/molecular mechanics, molecular dynamics simulations, and potential of mean force were employed to study the interactions established between GlcNAcstatin and a bacterial O-GlcNAcase enzyme from Clostridium perfringens. The results reveal that the imidazole nitrogen atom of GlcNAcstatin has shown a better interaction with the active site of Clostridium perfringens in its protonated form, which is compatible with a substrate-assisted reaction mechanism involving two conserved aspartate residues (297 and 298). Furthermore, the quantum mechanics/molecular mechanics-molecular dynamics simulations appointed a strong interaction between Asp401, Asp298, and Asp297 residues and the GlcNAcstatin inhibitor, which is in accordance with experimental data. Lastly, these results may contribute to understand the molecular mechanism of inhibition of Clostridium perfringens by GlcNAcstatin inhibitor and, consequently, this study might be useful to design new molecules with more interesting inhibitory activity.
Asunto(s)
Acetilglucosamina/análogos & derivados , Acetilglucosamina/química , Imidazoles/química , beta-N-Acetilhexosaminidasas/química , Acetilglucosamina/farmacología , Dominio Catalítico , Clostridium perfringens/efectos de los fármacos , Clostridium perfringens/enzimología , Imidazoles/farmacología , Ligandos , Modelos Moleculares , Simulación de Dinámica Molecular , Termodinámica , beta-N-Acetilhexosaminidasas/antagonistas & inhibidores , beta-N-Acetilhexosaminidasas/metabolismoRESUMEN
The aim of the present study was to determine whether dehydroleucodine, xanthatin and 3-benzyloxymethyl-5H-furan-2-one inhibit the activation of human leukemic LAD2 mast cells induced by compound 48/80 or the calcium ionophore A23187. LAD2 cells were preincubated in the presence of test drugs and then challenged with the secretagogues. This study provides the first evidence in favor of the view that dehydroleucodine and xanthatin inhibit the degranulation of LAD2 cells, thus acting as human mast cell stabilizers. These molecules could be effective in the treatment of human diseases associated with inappropriate mast cell activation.
Asunto(s)
Furanos/farmacología , Lactonas/farmacología , Mastocitos/metabolismo , Sesquiterpenos/farmacología , Calcimicina/farmacología , Ionóforos de Calcio/farmacología , Degranulación de la Célula/efectos de los fármacos , Línea Celular Tumoral , Humanos , Cinética , Leucemia de Mastocitos/metabolismo , Leucemia de Mastocitos/patología , Mastocitos/fisiología , Mastocitos/ultraestructura , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , beta-N-Acetilhexosaminidasas/metabolismo , p-Metoxi-N-metilfenetilamina/farmacologíaRESUMEN
From cultures of thermophilic soil fungus Humicola grisea var thermoidea, a δ-lactam derivative (3-(2-(4-hydroxyphenyl)-2-oxoethyl)-5,6-dihydropyridin-2(1H)-one) that displayed anti-allergic activity was isolated, which was predicted by in silico computational chemistry approaches. The in vitro anti-allergic activity was investigated by ß-hexosaminidase release assay in rat basophilic leukaemia RBL-2H3 cells. The δ-lactam derivative exhibited similar anti-allergic activity (IC(50) = 18.7 ± 6.7 µM) in comparison with ketotifen fumarate (IC(50) = 15.0 ± 1.3 µM) and stronger anti-allergic activity than azelastine (IC(50) = 32.0 µM). Also, the MTT cytotoxicity assay with RBL-2H3 cells showed that δ-lactam does not display cytotoxicity at concentrations lower than 50 µM. This study suggests that the δ-lactam derivative has the potential to be used as a lead compound in the development of anti-allergic drugs for clinical use in humans.
Asunto(s)
Antialérgicos/química , Antialérgicos/farmacología , Ascomicetos/química , Lactamas/química , Piridonas/química , Piridonas/farmacología , Animales , Degranulación de la Célula/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Simulación por Computador , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos/métodos , Concentración 50 Inhibidora , Cetotifen/farmacología , Espectroscopía de Resonancia Magnética , Estructura Molecular , Ftalazinas/farmacología , Ratas , Microbiología del Suelo , beta-N-Acetilhexosaminidasas/metabolismoRESUMEN
O-Glycoprotein 2-acetamino-2-deoxy-ß-D-glucopyranosidase (O-GlcNAcase) hydrolyzes O-linked 2-acetamido-2-deoxy-ß-D-glucopyranoside (O-GlcNAc) residues from post-translationally modified serine/threonine residues of nucleocytoplasmic protein. The chemical process involves substrate-assisted catalysis, where two aspartate residues have been identified as the two key catalytic residues of O-GlcNAcase. In this report, the first step of the catalytic mechanism used by O-GlcNAcase involving substrate-assisted catalysis has been studied using a hybrid quantum mechanical/molecular mechanical (QM/MM) Molecular Dynamics (MD) calculations. The free energy profile shows that the formation of the oxazoline intermediate in the O-GlcNAcase catalytic reaction takes place by means of a stepwise mechanism. The first step would be a cyclization of the acetomide group, which seems to be dependent on the proton transfer from a conserved aspartate, Asp298 in Clostridium perfringens O-GlcNAcase. From this new intermediate, a proton is transferred from the azoline ring to another conserved aspartate (Asp297) thus forming the oxazoline ion and departure of the aglycone. In addition, averaged values of protein-substrate interaction energy along the reaction path shows that, in fact, the transition states present the highest binding affinities. A deeper analysis of the binding contribution of the individual residues shows that Asp297, Asp298, and Asp401 are basically responsible of the stabilization of these complexes. These results would explain why O-(2-acetamido-2deoxy-d-glucopyranosylidene)amino-N-phenycarbamate (PUGNAc), 1,2-dideoxy-2'-methyl-α-D-glucopyranoso-[2,1-d]-Δ2'-thiazoline (NAG-thiazoline), and GlcNAcstatin derivatives are potent inhibitors of this enzyme, resembling the two transition states of the O-GlcNAcase catalytic reaction path. These results may be useful to rational design compounds with more interesting inhibitory activity.
Asunto(s)
Clostridium perfringens/enzimología , beta-N-Acetilhexosaminidasas/antagonistas & inhibidores , beta-N-Acetilhexosaminidasas/metabolismo , Clostridium perfringens/química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Simulación de Dinámica Molecular , Oxazoles/metabolismo , Teoría Cuántica , Especificidad por Sustrato , Termodinámica , beta-N-Acetilhexosaminidasas/químicaRESUMEN
In a previous work, we have demonstrated that Minthostachys verticillata essential oil has immunomodulatory effects in vitro on cells from allergic patients. Here we characterized main components of M. verticillata essential oil and also tested if these compounds modulate In vitro and in vivo the immediate-type allergic reaction. Gas chromatography was used to identify main components of the essential oil. Pulegone (63.4â%), menthone (15.9â%), and limonene (2.1â%) were found as main classes. IL-13 levels were evaluated from lymphocytes cultures stimulated with allergen alone or combined with monoterpenes. All compounds stimulated cell proliferation but, interestingly, promoted a reduction of IL-13 values, limonene and the mixture of the three compounds being the most active. ß-Hexosaminidase release was determined from basophils to which essential oil or monoterpenes were added. We observed that, whichever combination of monoterpenes was used, ß-hexosaminidase release was diminished in all cases. Obtained values were even lower than those of antiallergic drug desloratadine. Essential oil and limonene inhibited mast cell activation and degranulation in the skin when testing passive cutaneous anaphylaxis, limonene being the most active. In conclusion, limonene was the compound that showed the most potent immunomodulatory activity. This fact suggests that it constitutes a promising natural alternative for a novel treatment of allergic diseases.
Asunto(s)
Antialérgicos/farmacología , Hipersensibilidad Inmediata/tratamiento farmacológico , Lamiaceae/química , Monoterpenos/farmacología , Aceites Volátiles/farmacología , Adolescente , Adulto , Animales , Antialérgicos/aislamiento & purificación , Basófilos/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Niño , Preescolar , Monoterpenos Ciclohexánicos , Ciclohexenos/aislamiento & purificación , Ciclohexenos/farmacología , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Lactante , Interleucina-13/análisis , Leucocitos Mononucleares/efectos de los fármacos , Limoneno , Masculino , Mastocitos/efectos de los fármacos , Mentol/aislamiento & purificación , Mentol/farmacología , Ratones , Ratones Endogámicos BALB C , Monoterpenos/aislamiento & purificación , Aceites Volátiles/química , Aceites Volátiles/aislamiento & purificación , Terpenos/aislamiento & purificación , Terpenos/farmacología , Adulto Joven , beta-N-Acetilhexosaminidasas/efectos de los fármacos , beta-N-Acetilhexosaminidasas/metabolismoRESUMEN
BACKGROUND: Dried blood spots (DBS) on filter paper is a valuable sampling technique in clinical chemistry, but the stability of enzymes used in the diagnosis of lysosomal storage diseases (LSDs) needs to be evaluated. METHODS: In a first experiment, blood from 20 subjects was collected using a syringe without additives and distributed into EDTA tubes, heparin tubes, and spotted on filter paper for the comparison of sampling effects. In a second experiment, blood from 30 healthy subjects was spotted on filter paper and analyzed for ß-galactosidase and total hexosaminidase activities after storage of the samples at different temperatures for up to 180 days. RESULTS: Initially, we observed that enzyme activities were the same, independent of the collection method. When DBS was stored at 37°C the activity of ß-galactosidase dropped to 85% of the initial value after 180 days (p<0.05). At all other temperatures (-20°C, 4°C and 25°C), the results were within the methodological error. Total hexosaminidase activity did not change significantly during the entire study period and at different storage temperatures. CONCLUSIONS: The two enzymes investigated in the present study may be stored for up to 17 days (ß-galactosidase) or 180 days (total hexosaminidase) until analysis without loss of activity.
Asunto(s)
Recolección de Muestras de Sangre/métodos , Temperatura , beta-Galactosidasa/metabolismo , beta-N-Acetilhexosaminidasas/metabolismo , Recolección de Muestras de Sangre/instrumentación , Humanos , Enfermedades por Almacenamiento Lisosomal/sangre , Enfermedades por Almacenamiento Lisosomal/enzimología , Papel , Factores de Tiempo , beta-Galactosidasa/sangre , beta-N-Acetilhexosaminidasas/sangreRESUMEN
The production of chitinases and hydrophobins from Lecanicillium lecanii was influenced by the cultivation method and type of carbon source. Crude enzyme obtained from solid-substrate culture presented activities of exochitinases (32 and 51 kDa), endochitinases (26 kDa), ß-N-acetylhexosaminidases (61, 80, 96 and 111 kDa). Additionally, submerged cultures produced exochitinases (32 and 45 kDa), endochitinases (10 and 26 kDa) and ß-N-acetylhexosaminidases (61, 96 and 111 kDa). ß-N-acetylhexosaminidases activity determined in solid-substrate culture with added chitin was ca. threefold (7.58 ± 0.57 U mg(-1)) higher than submerged culture (2.73 + 0.57 U mg(-1)). Similarly, hydrophobins displayed higher activities in solid-substrate culture (627.3 ± 2 µg protein mL(-1)) than the submerged one (57.4 ± 4.7 µg protein mL(-1)). Molecular weight of hydrophobins produced in solid-substrate culture was 7.6 kDa and they displayed surface activity on Teflon.
Asunto(s)
Quitinasas , Proteínas Fúngicas , Hypocreales/enzimología , Quitina/metabolismo , Quitinasas/química , Quitinasas/aislamiento & purificación , Quitinasas/metabolismo , Medios de Cultivo/química , Proteínas Fúngicas/química , Proteínas Fúngicas/aislamiento & purificación , Proteínas Fúngicas/metabolismo , Hexosaminidasas/aislamiento & purificación , Hexosaminidasas/metabolismo , Microscopía Electrónica de Rastreo , Politetrafluoroetileno/química , Esporas Fúngicas/crecimiento & desarrollo , Especificidad por Sustrato , beta-N-Acetilhexosaminidasas/aislamiento & purificación , beta-N-Acetilhexosaminidasas/metabolismoRESUMEN
The enzyme O-glycoprotein 2-acetamino-2-deoxy-beta-d-glucopyranosidase (O-GlcNAcase) is responsible for the removal of N-acetylglucosamine moieties from 2-acetamido-2-deoxy-beta-D-glucopyranose (O-GlcNAc) residues of serine/threonine residues of modified proteins. We herein present results of hybrid quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) simulations applied to the study of the interactions established between a bacterial Clostridium perfringens homologue (CpNagJ) and PUGNAc, a potent known inhibitor of this enzyme. Electrostatic binding free energy and energy term decomposition have been computed for the wild-type CpNagJ and several mutants: D297N, D298N, Y335F, N390A, N396A, D401A, and W490A. The theoretical results have been compared with recently experimental data based on crystallographic and mutation studies on the same system. Our results reveal that, first, there is a strong interaction between Asp401, Asp298, and Asp297 residues and the PUGNAc inhibitor; and, second, the electrostatic substrate binding free energy is higher in wild-type, N390A and W490A mutants than in D297N, D298N, Y335F, N396A, and D401A ones, in accordance with the experimental results. Finally, both our theoretical predictions and the experimental data are compatible with a substrate-assisted reaction mechanism, involving two conserved aspartate residues.