RESUMEN
The discovery of giant viruses in the last years has fascinated the scientific community due to virus particles size and genome complexity. Among such fantastic discoveries, we have recently described tupanviruses, which particles present a long tail, and has a genome that contains the most complete set of translation-related genes ever reported in the known virosphere. Here we describe a new kind of virus-host interaction involving tupanvirus. We observed that tupanvirus-infected amoebas were induced to aggregate with uninfected cells, promoting viral dissemination and forming giant host cell bunches. Even after mechanical breakdown of bunches, amoebas reaggregated within a few minutes. This remarkable interaction between infected and uninfected cells seems to be promoted by the expression of a mannose receptor gene. Our investigations demonstrate that the pre-treatment of amoebas with free mannose inhibits the formation of bunches, in a concentration-dependent manner, suggesting that amoebal-bunch formation correlates with mannose receptor gene expression. Finally, our data suggest that bunch-forming cells are able to interact with uninfected cells promoting the dissemination and increase of tupanvirus progeny.
Asunto(s)
Amoeba/virología , Agregación Celular/efectos de los fármacos , Virus Gigantes/patogenicidad , Interacciones Huésped-Patógeno , Virosis/transmisión , Amoeba/citología , Virus Gigantes/genética , Lectinas Tipo C/metabolismo , Manosa/farmacología , Receptor de Manosa , Lectinas de Unión a Manosa/metabolismo , Receptores de Superficie Celular/metabolismoRESUMEN
In this work, we evaluated the ability of Punica granatum sarcotesta lectin (PgTeL) to impair the growth and viability of the Staphylococcus aureus clinical isolates 8325-4 (non-resistant) and LAC USA300 (MRSA strain). The effects of this lectin on aggregating, hemolytic activity, biofilm-forming ability, and expression of virulence genes (hla, rnaIII, and spa) were also investigated. PgTeL showed antibacterial activity against 8325-4 and LAC USA300 strains by interfering with both the growth (MIC50 of 6.25 and 12.5⯵g/mL, respectively) and survival (MBC values of 25.0 and 50.0⯵g/mL, respectively). Culture growth started only at the ninth (8325-4) and tenth (LAC USA300) hour in the presence of PgTeL at MIC50, while growth was detected since the first hour in the control. The lectin caused markedly altered cell morphology in both the strains. Although, at the MIC50, PgTeL caused structural alterations, most cells were still viable, while at the MBC it promoted cell injury and death. PgTeL showed anti-aggregation effect and exhibited antibiofilm activity against both the isolates. However, the lectin did not interfere with the hemolytic activity of LAC USA300 and with the expression of hla, rnaIII, and spa genes. In conclusion, PgTeL is a lectin with multiple inhibitory effects on S. aureus clinical isolates.
Asunto(s)
Biopelículas/efectos de los fármacos , Lectinas/química , Lythraceae/química , Staphylococcus aureus/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Agregación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Humanos , Lectinas/farmacología , Staphylococcus aureus/patogenicidadRESUMEN
PURPOSE: The effect of a prophylactic oleuropein-rich diet before anesthesia accompanied by the widely-used steroid-based neuromuscular drug rocuronium on mast cell activation was investigated in the study. METHODS: 14 rabbits used in the study. The rabbits in the oleuropein group were given oleuropein-rich extract added to the animals' water at doses of 20 mg/kg oleuropein for 15 days orally. After 15 days, all rabbits in the two groups were given general anesthesia with rocuronium of 1 mg/kg. After 1 day, animals were sacrificed and the liver tissue sections stained with H&E, toluidine blue and tryptase for immunohistochemical study. RESULTS: There was no statistically significant difference between ALT, AST and albumin averages of the oleuropein and control groups (p> 0.05). The tryptase average of the control group was higher than the tryptase average of the oleuropein group and this difference was statistically significant (p=0.003). The T. blue average in the oleuropein group was higher than the control group. However, there was no statistically significant difference between groups (p=0.482). CONCLUSIONS: Rocuronium adverse effects, like hypersensitivity and anaphylaxis, may limit routine use of this substance. The use of oleuropein reduced the number of inflammatory cells and prevented degranulation.
Asunto(s)
Anestesia General/efectos adversos , Antiinflamatorios/administración & dosificación , Iridoides/administración & dosificación , Mastocitos/efectos de los fármacos , Fármacos Neuromusculares no Despolarizantes/efectos adversos , Rocuronio/efectos adversos , Alanina Transaminasa/sangre , Animales , Aspartato Aminotransferasas/sangre , Agregación Celular/efectos de los fármacos , Degranulación de la Célula/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Dietoterapia/métodos , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/prevención & control , Inmunohistoquímica , Glucósidos Iridoides , Hígado/efectos de los fármacos , Hígado/enzimología , Masculino , Mastocitos/patología , Profilaxis Pre-Exposición/métodos , Conejos , Distribución Aleatoria , Reproducibilidad de los Resultados , Albúmina Sérica/análisisRESUMEN
Abstract Purpose: The effect of a prophylactic oleuropein-rich diet before anesthesia accompanied by the widely-used steroid-based neuromuscular drug rocuronium on mast cell activation was investigated in the study. Methods: 14 rabbits used in the study. The rabbits in the oleuropein group were given oleuropein-rich extract added to the animals' water at doses of 20 mg/kg oleuropein for 15 days orally. After 15 days, all rabbits in the two groups were given general anesthesia with rocuronium of 1 mg/kg. After 1 day, animals were sacrificed and the liver tissue sections stained with H&E, toluidine blue and tryptase for immunohistochemical study. Results: There was no statistically significant difference between ALT, AST and albumin averages of the oleuropein and control groups (p> 0.05). The tryptase average of the control group was higher than the tryptase average of the oleuropein group and this difference was statistically significant (p=0.003). The T. blue average in the oleuropein group was higher than the control group. However, there was no statistically significant difference between groups (p=0.482). Conclusions: Rocuronium adverse effects, like hypersensitivity and anaphylaxis, may limit routine use of this substance. The use of oleuropein reduced the number of inflammatory cells and prevented degranulation.
Asunto(s)
Animales , Masculino , Conejos , Fármacos Neuromusculares no Despolarizantes/efectos adversos , Iridoides/administración & dosificación , Rocuronio/efectos adversos , Anestesia General/efectos adversos , Mastocitos/efectos de los fármacos , Antiinflamatorios/administración & dosificación , Aspartato Aminotransferasas/sangre , Albúmina Sérica/análisis , Distribución Aleatoria , Degranulación de la Célula/efectos de los fármacos , Agregación Celular/efectos de los fármacos , Reproducibilidad de los Resultados , Cromatografía Líquida de Alta Presión , Dietoterapia/métodos , Alanina Transaminasa/sangre , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/prevención & control , Profilaxis Pre-Exposición/métodos , Hígado/efectos de los fármacos , Hígado/enzimología , Mastocitos/patologíaRESUMEN
There is no consensus on aspects of equine bone marrow collection and processing. The study aimed to describe the collection of large volumes of bone marrow from horses of advanced age, with emphasis on bone marrow mononuclear cells (BMMCs) recovery and viability after cryopreservation. Fourteen horses, aged 3-24 years, were divided into three experiments. E1 studied the feasibility of collecting 200 mL from the sternums of horses of advanced age; E2 examined the number of cells obtained from the first and last syringe of each puncture; and E3 investigated the influence of heparin concentration on the prevention of cell aggregation, and cell viability after freezing in liquid nitrogen. Bone marrow aspirations were done with syringes pre-filled with Iscove's modified Dulbecco's medium and different concentrations of sodium heparin. BMMCs were counted, cell viability was determined, and samples were frozen. Bone marrow collection from the sternum is safe, even at large volumes and from horses of advanced age, and the number of cells recovered decreases with successive aspirations (p < 0.0001). Heparin concentration influenced cell aggregation, and recovered cells continued to be commercially viable after 150 days in frozen storage.
Asunto(s)
Células de la Médula Ósea/fisiología , Agregación Celular/efectos de los fármacos , Criopreservación/métodos , Heparina/farmacología , Leucocitos Mononucleares/fisiología , Animales , Recuento de Células , Supervivencia Celular/efectos de los fármacos , Femenino , Congelación , Caballos , Masculino , Esternón/citologíaRESUMEN
Trichomonas vaginalis is a common sexually transmitted parasite that colonizes the human urogenital tract. Infections range from asymptomatic to highly inflammatory, depending on the host and the parasite strain. Different T. vaginalis strains vary greatly in their adherence and cytolytic capacities. These phenotypic differences might be attributed to differentially expressed genes as a consequence of extra-genetic variation, such as epigenetic modifications. In this study, we explored the role of histone acetylation in regulating gene transcription and pathogenesis in T. vaginalis. Here, we show that histone 3 lysine acetylation (H3KAc) is enriched in nucleosomes positioned around the transcription start site of active genes (BAP1 and BAP2) in a highly adherent parasite strain; compared with the low acetylation abundance in contrast to that observed in a less-adherent strain that expresses these genes at low levels. Additionally, exposition of less-adherent strain with a specific histone deacetylases inhibitor, trichostatin A, upregulated the transcription of BAP1 and BAP2 genes in concomitance with an increase in H3KAc abundance and chromatin accessibility around their transcription start sites. Moreover, we demonstrated that the binding of initiator binding protein, the transcription factor responsible for the initiation of transcription of ~75% of known T. vaginalis genes, depends on the histone acetylation state around the metazoan-like initiator to which initiator binding protein binds. Finally, we found that trichostatin A treatment increased parasite aggregation and adherence to host cells. Our data demonstrated for the first time that H3KAc is a permissive histone modification that functions to mediate both transcription and pathogenesis of the parasite T. vaginalis.
Asunto(s)
Adhesión Celular/efectos de los fármacos , Agregación Celular/efectos de los fármacos , Histonas/metabolismo , Vaginitis por Trichomonas/patología , Trichomonas vaginalis/genética , Trichomonas vaginalis/patogenicidad , Acetilación/efectos de los fármacos , Adhesión Celular/genética , Adhesión Celular/fisiología , Agregación Celular/fisiología , Línea Celular Tumoral , Cuello del Útero/citología , Cuello del Útero/metabolismo , Cuello del Útero/parasitología , Cromatina/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Regulación de la Expresión Génica , Células HeLa , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Ácidos Hidroxámicos/farmacología , Metaloendopeptidasas/genética , Unión Proteica/fisiología , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Transcripción Genética/genética , Activación Transcripcional/genética , Vaginitis por Trichomonas/parasitología , Trichomonas vaginalis/metabolismoRESUMEN
Canavalia ensiformis (ConA), Canavalia brasiliensis (Conbr), and Cratylia floribunda (CFL) lectins have exhibited glucose-mannose binding specificity. We investigated the effect of fetal bovine serum (FBS) concentrations (1, 5, 10, and 20%) on the cytotoxic effect of these lectins against breast tumor cell line MCF-7. Cell viability was examined using the MTT reduction assay. When cells were grown in a medium supplemented with a higher serum concentration (10 or 20%), all lectins were much less toxic. When we used 1% FBS, it was possible to achieve a concentration-dependent activity by all examined lectins, with an IC(50) of 3.5, 25, and 60 µg/mL for ConA, Conbr, and CFL, respectively. All lectins incubated with 1% FBS induced apoptosis and DNA damage in MCF-7 cells. We conclude that ConA, Conbr, and CFL lectins' cytotoxic and genotoxic effects were observed only at low concentrations of serum.
Asunto(s)
Apoptosis/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Canavalia/química , Lectinas de Plantas/farmacología , Suero/química , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Bovinos , Agregación Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Feto , Glucosa/metabolismo , Humanos , Manosa/metabolismo , Pruebas de Mutagenicidad , Lectinas de Plantas/química , Lectinas de Plantas/aislamiento & purificación , Unión Proteica , Semillas/químicaRESUMEN
Connective tissue growth factor (CCN2/CTGF) is a matricellular-secreted protein involved in extracellular matrix remodeling. The P19 cell line is an embryonic carcinoma line widely used as a cellular model for differentiation and migration studies. In the present study, we employed an exogenous source of CCN2 and small interference RNA to address the role of CCN2 in the P19 cell aggregation phenomenon. Our data showed that increasing CCN2 protein concentrations from 0.1 to 20 nM decreased the number of cell clusters and dramatically increased cluster size without changing proliferation or cell survival, suggesting that CCN2 induced aggregation. In addition, CCN2 specific silencing inhibited typical P19 cell aggregation, which could be partially rescued by 20 nM CCN2. The present study demonstrates that CCN2 is a key molecule for cell aggregation of embryonic P19 cells.
Asunto(s)
Humanos , Agregación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Factor de Crecimiento del Tejido Conjuntivo/farmacología , Células Madre de Carcinoma Embrionario/efectos de los fármacos , Adhesión Celular , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiologíaRESUMEN
Connective tissue growth factor (CCN2/CTGF) is a matricellular-secreted protein involved in extracellular matrix remodeling. The P19 cell line is an embryonic carcinoma line widely used as a cellular model for differentiation and migration studies. In the present study, we employed an exogenous source of CCN2 and small interference RNA to address the role of CCN2 in the P19 cell aggregation phenomenon. Our data showed that increasing CCN2 protein concentrations from 0.1 to 20 nM decreased the number of cell clusters and dramatically increased cluster size without changing proliferation or cell survival, suggesting that CCN2 induced aggregation. In addition, CCN2 specific silencing inhibited typical P19 cell aggregation, which could be partially rescued by 20 nM CCN2. The present study demonstrates that CCN2 is a key molecule for cell aggregation of embryonic P19 cells.
Asunto(s)
Agregación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Factor de Crecimiento del Tejido Conjuntivo/farmacología , Células Madre de Carcinoma Embrionario/efectos de los fármacos , Adhesión Celular , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , HumanosRESUMEN
Taenia crassiceps cysticerci (cysts) reproduce by budding. The cysts' production of buds was measured in vitro to explore parasite and environmental-related factors involved in the extreme individual variation in parasite loads of inbred mice. Cysts were placed in in vitro culture for 10 days at initial parasite densities of 1, 5, 10 cysts/well in 1 ml of RPMI Medium 1640 without serum. Results showed that there is considerable intrinsic initial variation among inoculated cysts in their production of buds and that increasing parasite density (crowding) stimulates the overall production of buds and recruit into budding most of the cysts. Identical cultures were then subjected to various treatments such as heating and exposure to peroxide to induce stress, or to 17beta-estradiol, insulin, glucose, or insulin+glucose to supplement putatively limiting hormonal and energy resources. All treatments increased budding but the parasites' strong budding response to crowding alone overshadows the other treatments.
Asunto(s)
Cysticercus/citología , Cysticercus/fisiología , Metabolismo Energético/efectos de los fármacos , Hormonas/farmacología , Transducción de Señal/efectos de los fármacos , Estrés Fisiológico/efectos de los fármacos , Animales , Agregación Celular/efectos de los fármacos , Cysticercus/efectos de los fármacos , Parásitos/citología , Parásitos/efectos de los fármacosRESUMEN
The effects of physalin B (a natural secosteroidal chemical from Physalis angulata, Solanaceae) on phagocytosis and microaggregation by hemocytes of 5th-instar larvae of Rhodnius prolixus were investigated. In this insect, hemocyte phagocytosis and microaggregation are known to be induced by the platelet-activating factor (PAF) or arachidonic acid (AA) and regulated by phospholipase A(2) (PLA(2)) and PAF-acetyl hydrolase (PAF-AH) activities. Phagocytic activity and formation of hemocyte microaggregates by Rhodnius hemocytes were strongly blocked by oral treatment of this insect with physalin B (1mug/mL of blood meal). The inhibition induced by physalin B was reversed for both phagocytosis and microaggregation by exogenous arachidonic acid (10microg/insect) or PAF (1microg/insect) applied by hemocelic injection. Following treatment with physalin B there were no significant alterations in PLA(2) activities, but a significant enhancement of PAF-AH was observed. These results show that physalin B inhibits hemocytic activity by depressing insect PAF analogous (iPAF) levels in hemolymph and confirm the role of PAF-AH in the cellular immune reactions in R. prolixus.
Asunto(s)
1-Alquil-2-acetilglicerofosfocolina Esterasa/metabolismo , Hemocitos/inmunología , Proteínas de Insectos/metabolismo , Fagocitosis/efectos de los fármacos , Rhodnius/enzimología , Secoesteroides/farmacología , Animales , Ácido Araquidónico/farmacología , Agregación Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Hemocitos/efectos de los fármacos , Hemocitos/enzimología , Hemocitos/microbiología , Factor de Activación Plaquetaria/farmacología , Rhodnius/efectos de los fármacos , Rhodnius/inmunología , Rhodnius/microbiología , Saccharomyces cerevisiae/fisiologíaRESUMEN
The tumor necrosis factor alpha (TNFalpha) plays a dual role in producing either neurodegeneration or neuroprotection in the central nervous system. Despite that TNFalpha was initially described as a cell death inductor, neuroprotective effects against cell death induced by several neurotoxic insults have been reported. Tau hyperphosphorylation and neuronal death found in Alzheimer disease is mediated by deregulation of the cdk5/p35 complex induced by Abeta treatments. Since TNFalpha affects cdk5 activity, we investigated its possible protective role against the Abeta-induced neurodegeneration, as mediated by cdk5. TNFalpha pretreatments significantly reduced the hippocampal neuronal cell death induced by the effects of Abeta(42) peptide. In addition, this pretreatment reduced the increase in the activity of cdk5 induced by Abeta(42) in primary neurons. Next, we investigated the Alzheimer type phosphorylation of tau protein induced by Abeta(42). We observed that the pretreatment of neurons with TNFalpha reduces tau hyperphosphorylation. Taken together, these results define a novel neuroprotective effect of TNFalpha in preventing neuronal cell death and cdk5-dependent tau hyperphosphorylation. This phenomenon, taken together with other previous findings, suggests that the inflammatory response due to Abeta peptide plays a key role in the development of Alzheimer etiopathogenesis.
Asunto(s)
Péptidos beta-Amiloides/toxicidad , Quinasa 5 Dependiente de la Ciclina/metabolismo , Fármacos Neuroprotectores/farmacología , Neurotoxinas/toxicidad , Fragmentos de Péptidos/toxicidad , Factor de Necrosis Tumoral alfa/farmacología , Enfermedad de Alzheimer/patología , Animales , Agregación Celular/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Corteza Cerebral/citología , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/embriología , Quinasa 5 Dependiente de la Ciclina/genética , Hipocampo/citología , Hipocampo/efectos de los fármacos , Hipocampo/enzimología , Humanos , Mitocondrias/efectos de los fármacos , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/enzimología , Fosforilación/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Proteínas tau/metabolismoRESUMEN
Galectins, a family of structurally related carbohydrate-binding proteins, contribute to different events associated with cancer biology, including apoptosis, homotypic cell aggregation, angiogenesis and tumor-immune escape. To interfere with galectin-carbohydrate interactions during tumor progression, a current challenge is the design of specific galectin inhibitors for therapeutic purposes. Here, we report the synthesis of three novel low molecular weight synthetic lactulose amines (SLA): (1) N-lactulose-octamethylenediamine (LDO), (2) N,N'-dilactulose-octamethylenediamine (D-LDO), and (3) N,N'-dilactulose-dodecamethylenediamine (D-LDD). These compounds showed a differential ability to inhibit binding of galectin-1 and/or galectin-3 to the highly glycosylated protein 90K in solid-phase assays. In addition, each compound demonstrated selective regulatory effects in different events linked to tumor progression including tumor-cell apoptosis, homotypic cell aggregation, and endothelial cell morphogenesis. Our results suggest that galectin inhibitors with subtle differences in their carbohydrate structures may be potentially used to specifically block different steps of tumor growth and metastasis.
Asunto(s)
Aminas/síntesis química , Aminas/farmacología , Antineoplásicos/síntesis química , Apoptosis/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Galectinas/antagonistas & inhibidores , Lactulosa/química , Aminas/sangre , Aminas/química , Antineoplásicos/química , Antineoplásicos/clasificación , Antineoplásicos/farmacología , Agregación Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Células Cultivadas , Células Endoteliales/citología , Galectinas/farmacología , Glicosilación , Humanos , Estructura MolecularRESUMEN
Physalins are seco-steroids obtained from plants of the family Solanaceae. Herein, we tested Physalis angulata L purified physalin B as an immunomodulatory compound in 5th-instar larvae of Rhodnius prolixus, which were systemically infected with the H14 Trypanosoma rangeli strain protozoan. In uninfected insects, the effective concentration of physalin B, which inhibited 50% of the blood ingested (ED(50)) volume, was 15.2+/-1.6 microg/ml of the meal. Ecdysis processes and mortality in uninfected larvae, treated orally with physalin B in concentrations ranging from 1 to 10 microg/ml, was similar to that observed in insects not treated with physalin B. However, R. prolixus larvae previously fed on blood containing 1.0, 0.1, and 0.01 microg of physalin B/ml exhibited mortality rates of 78.1, 54.3, and 12.7%, respectively, 6 days after inoculation of T. rangeli (1 x 10(3) parasites/insect), whereas only 7.2% mortality was observed in the control group, injected with sterile culture medium. The insects treated with physalin B (0.1 microg/ml) and inoculated with T. rangeli did not modify the phenoloxidase (PO) activity and total hemocyte count in the hemolymph. However, physalin B treatment caused a reduction in hemocyte micro-aggregation and nitric oxide production and enhanced the parasitemia in the hemolymph. These results demonstrate that physalin B from P. angulata is a potent immunomodulatory substance for the bloodsucking insect, R. prolixus.
Asunto(s)
Lactonas/farmacología , Rhodnius/inmunología , Esteroides/farmacología , Trypanosoma/inmunología , Animales , Catecol Oxidasa/metabolismo , Agregación Celular/efectos de los fármacos , Recuento de Células , Activación Enzimática/efectos de los fármacos , Precursores Enzimáticos/metabolismo , Hemocitos/citología , Hemocitos/efectos de los fármacos , Lactonas/química , Larva/efectos de los fármacos , Larva/inmunología , Larva/parasitología , Nitratos/metabolismo , Nitritos/metabolismo , Rhodnius/efectos de los fármacos , Rhodnius/parasitología , Secoesteroides , Esteroides/químicaRESUMEN
During development of the nervous system, neuronal precursors that originated in proliferative regions migrate along radial glial fibers to reach their final destination. P19 embryonal carcinoma (EC) stem cells exposed to retinoic acid (RA) differentiate into neurons, glia, and fibroblast-like cells. In this work, we induced P19 aggregates for 4 days with RA and plated them onto tissue culture dishes coated with poly-L-lysine. Several cells migrated out of and/or extended processes from the aggregates after 24 hr. Some cell processes were morphologically similar to radial glial fibers and stained for glial fibrillar acidic protein (GFAP) and nestin. Large numbers of migrating cells showed characteristics similar to those of bipolar migrating neurons and expressed the neuronal marker microtubule-associated protein 2. Furthermore, scanning electron microscopy analysis revealed an intimate association between the radial fibers and the migrating cells. Therefore, the migration of neuron-like cells on radial glia fibers in differentiated P19 aggregates resembled some of the migration models used thus far to study gliophilic neuronal migration. In addition, HPTLC analysis in this system showed the expression of 9-O-acetyl GD3, a ganglioside that has been associated with neuronal migration. Antibody perturbation assays showed that immunoblockage of 9-O-acetyl GD3 arrested neuronal migration in a reversible manner. In summary, we have characterized a new cell culture model for investigation of glial-guided neuronal migration and have shown that 9-O-acetyl GD3 ganglioside has an important role in this phenomenon.
Asunto(s)
Movimiento Celular/fisiología , Gangliósidos/metabolismo , Fibras Nerviosas/fisiología , Neuroglía/fisiología , Células Madre Pluripotentes/fisiología , Animales , Agregación Celular/efectos de los fármacos , Agregación Celular/fisiología , Técnicas de Cultivo de Célula , Diferenciación Celular/fisiología , Línea Celular Tumoral , Células Madre de Carcinoma Embrionario , Proteína Ácida Fibrilar de la Glía/metabolismo , Ratones , Ratones Endogámicos C3H , Células Madre Neoplásicas/citología , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/fisiología , Neuroglía/citología , Neuronas/citología , Neuronas/fisiología , Células Madre Pluripotentes/citología , Tretinoina/farmacologíaRESUMEN
Hemocoelic inoculation of epimastigotes of Trypanosoma rangeli strain H14 into 5th-instar larvae of Rhodnius prolixus previously fed on blood containing the same parasites, showed reduced number of hemocyte microaggregates in the hemolymph, enhanced number of flagellates in the hemolymph as well as increased mortality of these insects. All these effects were counteracted by combined inoculation of R. prolixus with T. rangeli and arachidonic acid. In vitro assays using hemolymph taken from insects previously fed on blood containing parasites showed that hemocyte microaggregation reactions were also attenuated when T. rangeli is used as inducer of the reaction, and that simultaneous applying T. rangeli with arachidonic counteracted the hemocyte microaggregation inhibition. We suggest that arachidonic acid pathway can be a mediator of hemocyte microaggregation reactions in the hemolymph of insects inoculated with T. rangeli, and that oral infection with this protozoan inhibits the release of arachidonic acid.
Asunto(s)
Ácido Araquidónico/metabolismo , Hemocitos/metabolismo , Insectos Vectores/parasitología , Rhodnius/parasitología , Trypanosoma/fisiología , Animales , Ácido Araquidónico/farmacología , Agregación Celular/efectos de los fármacos , Hemocitos/efectos de los fármacos , Hemocitos/inmunología , Hemolinfa/citología , Hemolinfa/inmunología , Hemolinfa/parasitología , Humanos , Inmunidad Celular , Insectos Vectores/citología , Insectos Vectores/inmunología , Larva/citología , Larva/inmunología , Larva/parasitología , Rhodnius/citología , Rhodnius/inmunología , Trypanosoma/inmunologíaRESUMEN
The epigenetic manipulation of precursors may provide data to elucidate the potential interactions among these cells in different brain regions. However, the response to epigenetic signals is modulated by the environment in which the cells are manipulated. Therefore, data regarding the action of a particular factor must be considered in the light of a specific system. To compare septal and striatal precursors, we have tested the effect of nerve growth factor (NGF) on the proliferation and neuronal differentiation of epidermal growth factor (EGF)-responsive cells from these brain regions. Precursors were cultivated as 'neuropheres' in serum free medium (SFM) to which NGF was added. NGF did not support the proliferation of EGF-generated precursors so that no differences in the cell magnitude with respect to control cultures were observed. Differentiation of precursors in SFM plus 1% fetal bovine serum (FBS) on poly-D-lysine showed that the neuron number was increased two-fold in septal cultures treated with NGF but not in those from striatum. A quantitative evaluation of the soma surface and the number of primary neurites showed differences between both populations of precursor-generated neurons. In addition, we also observed no influence of NGF on these parameters of cellular morphology. Thus, taken together these results seem to indicate that at this developmental stage in which these populations of precursors were isolated, heterogeneities exist between them, which is probably related to their origin and/or functional roles in vivo.
Asunto(s)
Cuerpo Estriado/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Factor de Crecimiento Nervioso/metabolismo , Neuronas/metabolismo , Tabique del Cerebro/metabolismo , Células Madre/metabolismo , Animales , Agregación Celular/efectos de los fármacos , Agregación Celular/fisiología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , División Celular/efectos de los fármacos , División Celular/fisiología , Células Cultivadas , Cuerpo Estriado/citología , Cuerpo Estriado/embriología , Medio de Cultivo Libre de Suero , Factor de Crecimiento Nervioso/administración & dosificación , Neuronas/clasificación , Neuronas/efectos de los fármacos , Neuronas/fisiología , Ratas , Ratas Wistar , Valores de Referencia , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Tabique del Cerebro/citología , Tabique del Cerebro/embriología , Células Madre/clasificación , Células Madre/efectos de los fármacos , Células Madre/fisiologíaRESUMEN
The objective of the present study was to investigate the multicellular resistance of human hepatocarcinoma cells BEL-7402 to pharmorubicin. Cells (1 x 10(4)) and 200 microcarrier Cytodex-3 beads were seeded onto a 24-well plate and cultured in RPMI 1640 medium. After the formation of multicellular aggregates, morphology and cell viability were analyzed by scanning electron microscopy, transmission electron microscopy and flow cytometry, respectively. The IC50 was determined by flow cytometry and MTT assay after the cells cultured in aggregates and monolayers were treated with pharmorubicin. The culture products exhibited structural characteristics somewhat similar to those of trabecular hepatocarcinoma in vivo. Among the microcarriers, cells were organized into several layers. Intercellular spaces were 0.5-2.0 microm wide and filled with many microvilli. The percent of viable cells was 87%. The cells cultured as multicellular aggregates were resistant to pharmorubicin with IC50 4.5-fold and 7.7-fold that of monolayer culture as determined by flow cytometry and MTT assay, respectively. This three-dimensional culture model may be used to investigate the mechanisms of multicellular drug resistance of hepatocarcinoma and to screen new anticancer drugs.
Asunto(s)
Antibióticos Antineoplásicos/farmacología , Carcinoma Hepatocelular/tratamiento farmacológico , Epirrubicina/farmacología , Neoplasias Hepáticas/tratamiento farmacológico , Esferoides Celulares/efectos de los fármacos , Antineoplásicos/farmacología , Agregación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Resistencia a Antineoplásicos , Citometría de Flujo/métodos , Humanos , Masculino , Microscopía Electrónica/métodos , Microscopía Electrónica de Rastreo/métodos , Persona de Mediana Edad , Esferoides Celulares/ultraestructura , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
Bioassay-guided isolation of Duguetia hadrantha yielded two new 4,5-dioxo-1-azaaporphinoids, hadranthine A (1) and hadranthine B (2), together with the known alkaloids imbiline-1 (3), sampangine (4), and 3-methoxysampangine (5), whose structures were determined primarily from 2D-NMR 1H-13C HMBC, and 1H-15N HMBC experiments. This is the first report of the co-occurrence of the copyrine alkaloids 4 and 5, as well as the first report of either copyrine or imbiline type alkaloids from a Duguetia species. Compounds 1, 4, and 5 demonstrated in vitro antimalarial activity against Plasmodium falciparum (W-2 clone), while 2 was inactive. Instead, 2 showed in vitro cytotoxicity to selected human cancer cell lines (IC50 = 3-6 microg/mL against SK-MEL, KB, BT-549, and SK-OV-3), and 4 was also cytotoxic to human malignant melanoma (IC50 = 0.37 microg/mL). Sampangine (4) also inhibited cell aggregation with a MIC value of <0.15 microg/mL, while 3-methoxysampangine (5) was only weakly active.
Asunto(s)
Alcaloides/aislamiento & purificación , Antifúngicos/aislamiento & purificación , Antimaláricos/aislamiento & purificación , Antineoplásicos Fitogénicos/aislamiento & purificación , Plantas Medicinales/química , Alcaloides/farmacología , Animales , Antifúngicos/farmacología , Antimaláricos/farmacología , Antineoplásicos Fitogénicos/farmacología , Brasil , Adhesión Celular/efectos de los fármacos , Agregación Celular/efectos de los fármacos , Humanos , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Paraguay , Extractos Vegetales/química , Plasmodium falciparum/efectos de los fármacos , Espectrofotometría Ultravioleta , Células Tumorales CultivadasRESUMEN
OBJECTIVE AND DESIGN: To study the neutrophil migration and aggregation induced by euphorbin, a D-galactose binding lectin from Euphorbia milii var. milli latex. MATERIALS AND METHODS: Euphorbin-induced neutrophil migration was evaluated in vivo and in vitro, in the absence or presence of soluble D-galactose. Neutrophil aggregation induced in vitro by euphorbin was determined by light microscopy. RESULTS: The neutrophil migration inducing activity of euphorbin was dose-dependent and inhibited by soluble D-galactose. Neutrophil aggregation was rapidly reversed when provoked by 0.1 mg/ml euphorbin. In higher concentrations, euphorbin caused persistent and more extensive neutrophil aggregation. CONCLUSIONS: Euphorbin induced neutrophil migration through its sugar recognition property. The transitory neutrophil aggregation, induced by a euphorbin quantity similar to that able to cause maximal chemotactic response, is characteristic of homotypic neutrophil adhesion, whereas persistent aggregation, provoked by higher euphorbin quantities, corresponds to cell agglutination by a multivalent lectin.