RESUMEN
Alcohol exposure can affect brain development, leading to long-lasting behavioral problems, including cognitive impairment, which together is defined as fetal alcohol spectrum disorder (FASD). However, the fundamental mechanisms through which this occurs are largely unknown. In this study, we report that the exposure of postnatal day 7 (P7) mice to ethanol activates caspase-3 via cannabinoid receptor type-1 (CB1R) in neonatal mice and causes a reduction in methylated DNA binding protein (MeCP2) levels. The developmental expression of MeCP2 in mice is closely correlated with synaptogenesis and neuronal maturation. It was shown that ethanol treatment of P7 mice enhanced Mecp2 mRNA levels but reduced protein levels. The genetic deletion of CB1R prevented, and administration of a CB1R antagonist before ethanol treatment of P7 mice inhibited caspase-3 activation. Additionally, it reversed the loss of MeCP2 protein, cAMP response element binding protein (CREB) activation, and activity-regulated cytoskeleton-associated protein (Arc) expression. The inhibition of caspase-3 activity prior to ethanol administration prevented ethanol-induced loss of MeCP2, CREB activation, epigenetic regulation of Arc expression, long-term potentiation (LTP), spatial memory deficits and activity-dependent impairment of several signaling molecules, including MeCP2, in adult mice. Collectively, these results reveal that the ethanol-induced CB1R-mediated activation of caspase-3 degrades the MeCP2 protein in the P7 mouse brain and causes long-lasting neurobehavioral deficits in adult mice. This CB1R-mediated instability of MeCP2 during active synaptic maturation may disrupt synaptic circuit maturation and lead to neurobehavioral abnormalities, as observed in this animal model of FASD.
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The present study was undertaken to evaluate the immediate and long-term effects of a single-day exposure to 5-Azacytidine (5-AzaC), a DNA methyltransferase inhibitor, on neurobehavioral abnormalities in mice. Our findings suggest that the 5-AzaC treatment significantly inhibited DNA methylation, impaired extracellular signal-regulated kinase (ERK1/2) activation and reduced expression of the activity-regulated cytoskeleton-associated protein (Arc). These events lead to the activation of caspase-3 (a marker for neurodegeneration) in several brain regions, including the hippocampus and cortex, two brain areas that are essential for memory formation and memory storage, respectively. 5-AzaC treatment of P7 mice induced significant deficits in spatial memory, social recognition, and object memory in adult mice and deficits in long-term potentiation (LTP) in adult hippocampal slices. Together, these data demonstrate that the inhibition of DNA methylation by 5-AzaC treatment in P7 mice causes neurodegeneration and impairs ERK1/2 activation and Arc protein expression in neonatal mice and induces behavioral abnormalities in adult mice. DNA methylation-mediated mechanisms appear to be necessary for the proper maturation of synaptic circuits during development, and disruption of this process by 5-AzaC could lead to abnormal cognitive function.
Asunto(s)
Azacitidina/toxicidad , Encefalopatías/inducido químicamente , Inhibidores Enzimáticos/toxicidad , Trastornos de la Memoria/etiología , Enfermedades Neurodegenerativas/inducido químicamente , Animales , Animales Recién Nacidos , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Metilación de ADN/efectos de los fármacos , Metilación de ADN/genética , Modelos Animales de Enfermedad , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/genética , Hipocampo/citología , Hipocampo/efectos de los fármacos , Potenciación a Largo Plazo/efectos de los fármacos , Potenciación a Largo Plazo/genética , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Enfermedades Neurodegenerativas/patología , Fosfopiruvato Hidratasa/metabolismo , Receptor Cannabinoide CB1/deficiencia , Receptor Cannabinoide CB1/genética , Transducción de Señal/efectos de los fármacos , Conducta SocialRESUMEN
RNA Polymerase II transcribes beyond what later becomes the 3' end of a mature messenger RNA (mRNA). The formation of most mRNA 3' ends results from pre-mRNA cleavage followed by polyadenylation. In vitro studies have shown that low concentrations of ATP stimulate the 3' cleavage reaction while high concentrations inhibit it, but the origin of these ATP effects is unknown. ATP might enable a cleavage factor kinase or activate a cleavage factor directly. To distinguish between these possibilities, we tested several ATP structural analogs in a pre-mRNA 3' cleavage reaction reconstituted from DEAE-fractionated cleavage factors. We found that adenosine 5'-(ß,γ-methylene)triphosphate (AMP-PCP) is an effective in vitro 3' cleavage inhibitor with an IC50 of â¼300 µM, but that most other ATP analogs, including adenosine 5'-(ß,γ-imido)triphosphate, which cannot serve as a protein kinase substrate, promoted 3' cleavage but less efficiently than ATP. In combination with previous literature data, our results do not support ATP stimulation of 3' cleavage through cleavage factor phosphorylation in vitro. Instead, the more likely mechanism is that ATP stimulates cleavage factor activity through direct cleavage factor binding. The mammalian 3' cleavage factors known to bind ATP include the cleavage factor II (CF IIm) Clp1 subunit, the CF Im25 subunit and poly(A) polymerase alpha (PAP). The yeast homolog of the CF IIm complex also binds ATP through yClp1. To investigate the mammalian complex, we used a cell-line expressing FLAG-tagged Clp1 to co-immunoprecipitate Pcf11 as a function of ATP concentration. FLAG-Clp1 co-precipitated Pcf11 with or without ATP and the complex was not affected by AMP-PCP. Diadenosine tetraphosphate (Ap4A), an ATP analog that binds the Nudix domain of the CF Im25 subunit with higher affinity than ATP, neither stimulated 3' cleavage in place of ATP nor antagonized ATP-stimulated 3' cleavage. The ATP-binding site of PAP was disrupted by site directed mutagenesis but a reconstituted 3' cleavage reaction containing a mutant PAP unable to bind ATP nevertheless underwent ATP-stimulated 3' cleavage. Fluctuating ATP levels might contribute to the regulation of pre-mRNA 3' cleavage, but the three subunits investigated here do not appear to be responsible for the ATP-stimulation of pre-mRNA cleavage.
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Adenosina Trifosfato/metabolismo , Precursores del ARN/metabolismo , Procesamiento Postranscripcional del ARN/fisiología , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Células HeLa , HumanosRESUMEN
The significant consequences of ethanol use during pregnancy are neurobehavioral abnormalities involving hippocampal and neocortex malfunctions that cause learning and memory deficits collectively named fetal alcohol spectrum disorder. However, the molecular mechanisms underlying these abnormalities are still poorly understood and therefore warrant systematic research. Here, we document novel epigenetic abnormalities in the mouse model of fetal alcohol spectrum disorder. Ethanol treatment of P7 mice, which induces activation of caspase 3, impaired DNA methylation through reduced DNA methyltransferases (DNMT1 and DNMT3A) levels. Inhibition of caspase 3 activity, before ethanol treatment, rescued DNMT1, DNMT3A proteins as well as DNA methylation levels. Blockade of histone methyltransferase (G9a) activity or cannabinoid receptor type-1 (CB1R), prior to ethanol treatment, which, respectively, inhibits or prevents activation of caspase 3, rescued the DNMT1 and DNMT3A proteins and DNA methylation. No reduction of DNMT1 and DNMT3A proteins and DNA methylation was found in P7 CB1R null mice, which exhibit no ethanol-induced activation of caspase 3. Together, these data demonstrate that ethanol-induced activation of caspase 3 impairs DNA methylation through DNMT1 and DNMT3A in the neonatal mouse brain, and such impairments are absent in CB1R null mice. Epigenetic events mediated by DNA methylation may be one of the essential mechanisms of ethanol teratogenesis. Schematic mechanism of action by which ethanol impairs DNA methylation. Studies have demonstrated that ethanol has the capacity to bring epigenetic changes to contribute to the development of fetal alcohol spectrum disorder (FASD). However, the mechanisms are not well studied. P7 ethanol induces the activation of caspase 3 and impairs DNA methylation through reduced DNA methyltransferases (DNMT1 and DNMT3A) proteins (â). The inhibition or genetic ablation of cannabinoid receptor type-1 or inhibition of histone methyltransferase (G9a) by Bix (-----) or inhibition of caspase 3 activation by Q- quinoline-Val-Asp(Ome)-CH2-O-phenoxy (Q-VD-OPh) () rescue loss of DNMT1, DNMT3A as well as DNA methylation. Hence, the putative DNMT1/DNMT3A/DNA methylation mechanism may have a potential regulatory role in FASD.
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ADN (Citosina-5-)-Metiltransferasas/biosíntesis , Metilación de ADN/efectos de los fármacos , Metilación de ADN/fisiología , Etanol/toxicidad , Receptor Cannabinoide CB1/deficiencia , Animales , Animales Recién Nacidos , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/antagonistas & inhibidores , ADN Metiltransferasa 3A , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
In rodents, many exogenous and endogenous cannabinoids, such as anandamide (AEA) and 2-arachidonyl glycerol (2-AG), have been shown to play an important role in certain hippocampal memory processes. However, the mechanisms by which endogenous AEA regulate this processes are not well understood. Here the effects of AEA on long-term potentiation (LTP), hippocampal-dependent learning and memory tasks, pERK1/2, pCaMKIV, and pCREB signaling events in both cannabinoid receptor type 1 (CB1R) wild-type (WT) and knockout (KO) mice were assessed following administration of URB597, an inhibitor of the fatty acid amide hydrolase (FAAH). Acute administration of URB597 enhanced AEA levels without affecting the levels of 2-AG or CB1R in the hippocampus and neocortex as compared to vehicle. In hippocampal slices, URB597 impaired LTP in CB1R WT but not in KO littermates. URB597 impaired object recognition, spontaneous alternation and spatial memory in the Y-maze test in CB1R WT mice but not in KO mice. Furthermore, URB597 enhanced ERK phosphorylation in WT without affecting total ERK levels in WT or KO mice. URB597 impaired CaMKIV and CREB phosphorylation in WT but not in KO mice. CB1R KO mice have a lower pCaMKIV/CaMKIV ratio and higher pCREB/CREB ratio as compared to WT littermates. Our results indicate that pharmacologically elevated AEA impair LTP, learning and memory and inhibit CaMKIV and CREB phosphorylation, via the activation of CB1Rs. Collectively, these findings also suggest that pharmacological elevation of AEA beyond normal concentrations is also detrimental for the underlying physiological responses.
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Ácidos Araquidónicos/fisiología , Endocannabinoides/fisiología , Aprendizaje/fisiología , Potenciación a Largo Plazo/fisiología , Memoria/fisiología , Receptor Cannabinoide CB1/fisiología , Amidohidrolasas/antagonistas & inhibidores , Animales , Benzamidas/farmacología , Proteína Quinasa Tipo 4 Dependiente de Calcio Calmodulina/fisiología , Carbamatos/farmacología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/fisiología , Glicéridos/fisiología , Sistema de Señalización de MAP Quinasas/fisiología , Masculino , Aprendizaje por Laberinto/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosforilación , Alcamidas Poliinsaturadas , Procesamiento Proteico-Postraduccional , Receptor Cannabinoide CB1/deficiencia , Receptor Cannabinoide CB1/genética , Memoria Espacial/fisiologíaRESUMEN
The 3' end formation of mammalian pre-mRNA contributes to gene expression regulation by setting the downstream boundary of the 3' untranslated region, which in many genes carries regulatory sequences. A large number of protein cleavage factors participate in this pre-mRNA processing step, but chemical tools to manipulate this process are lacking. Guided by a hypothesis that a PPM1 family phosphatase negatively regulates the 3' cleavage reaction, we have found a variety of new small molecule activators of the in vitro reconstituted pre-mRNA 3' cleavage reaction. New activators include a cyclic peptide PPM1D inhibitor, a dipeptide with modifications common to histone tails, abscisic acid and an improved l-arginine ß-naphthylamide analog. The minimal concentration required for in vitro cleavage has been improved from 200µM to the 200nM-100µM range. These compounds provide unexpected leads in the search for small molecule tools able to affect pre-mRNA 3' end formation.
Asunto(s)
Péptidos Cíclicos/farmacología , Procesamiento de Término de ARN 3'/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Células HeLa , Humanos , Estructura Molecular , Peso Molecular , Relación Estructura-ActividadRESUMEN
BACKGROUND: Ethanol exposure to rodents during postnatal day 7 (P7), which is comparable to the third trimester of human pregnancy, induces long-term potentiation and memory deficits. However, the molecular mechanisms underlying these deficits are still poorly understood. METHODS: In the present study, we explored the potential role of epigenetic changes at cannabinoid type 1 (CB1R) exon1 and additional CB1R functions, which could promote memory deficits in animal models of fetal alcohol spectrum disorder. RESULTS: We found that ethanol treatment of P7 mice enhances acetylation of H4 on lysine 8 (H4K8ace) at CB1R exon1, CB1R binding as well as the CB1R agonist-stimulated GTPγS binding in the hippocampus and neocortex, two brain regions that are vulnerable to ethanol at P7 and are important for memory formation and storage, respectively. We also found that ethanol inhibits cyclic adenosine monophosphate response element-binding protein (CREB) phosphorylation and activity-regulated cytoskeleton-associated protein (Arc) expression in neonatal and adult mice. The blockade or genetic deletion of CB1Rs prior to ethanol treatment at P7 rescued CREB phosphorylation and Arc expression. CB1R knockout mice exhibited neither ethanol-induced neurodegeneration nor inhibition of CREB phosphorylation or Arc expression. However, both neonatal and adult mice did exhibit enhanced CREB phosphorylation and Arc protein expression. P7 ethanol-treated adult mice exhibited impaired spatial and social recognition memory, which were prevented by the pharmacological blockade or deletion of CB1Rs at P7. CONCLUSIONS: Together, these findings suggest that P7 ethanol treatment induces CB1R expression through epigenetic modification of the CB1R gene, and that the enhanced CB1R function induces pCREB, Arc, spatial, and social memory deficits in adult mice.
Asunto(s)
Etanol/toxicidad , Regulación de la Expresión Génica/efectos de los fármacos , Trastornos de la Memoria/inducido químicamente , Enfermedades Neurodegenerativas/inducido químicamente , Receptor Cannabinoide CB1/efectos de los fármacos , Receptor Cannabinoide CB1/metabolismo , Complejo Relacionado con el SIDA/metabolismo , Acetilación/efectos de los fármacos , Factores de Edad , Animales , Animales Recién Nacidos/metabolismo , Animales Recién Nacidos/psicología , Proteína de Unión a CREB/metabolismo , Depresores del Sistema Nervioso Central/toxicidad , Epigénesis Genética/efectos de los fármacos , Exones/efectos de los fármacos , Femenino , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Histonas/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neocórtex/efectos de los fármacos , Neocórtex/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/psicología , Fosforilación/efectos de los fármacos , Receptor Cannabinoide CB1/deficiencia , Receptor Cannabinoide CB1/genética , Conducta Social , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Glycan array analysis of Sclerotium rolfsii lectin (SRL) revealed its exquisite binding specificity to the oncofetal Thomsen-Friedenreich (Galß1-3GalNAcα-O-Ser/Thr, T or TF) antigen and its derivatives. This study shows that SRL strongly inhibits the growth of human colon cancer HT29 and DLD-1 cells by binding to cell surface glycans and induction of apoptosis through both the caspase-8 and -9 mediated signaling. SRL showed no or very weak binding to normal human colon tissues but strong binding to cancerous and metastatic tissues. Intratumor injection of SRL at subtoxic concentrations in NOD-SCID mice bearing HT29 xenografts resulted in total tumor regression in 9 days and no subsequent tumor recurrence. As the increased expression of TF-associated glycans is commonly seen in human cancers, SRL has the potential to be developed as a therapeutic agent for cancer.
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Antígenos de Neoplasias/metabolismo , Antígenos de Carbohidratos Asociados a Tumores/metabolismo , Ascomicetos/química , Neoplasias del Colon/tratamiento farmacológico , Lectinas/uso terapéutico , Animales , Antígenos de Neoplasias/inmunología , Antígenos de Carbohidratos Asociados a Tumores/inmunología , Apoptosis/efectos de los fármacos , Caspasa 8/metabolismo , Caspasa 9/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias del Colon/inmunología , Neoplasias del Colon/metabolismo , Humanos , Inyecciones , Lectinas/aislamiento & purificación , Lectinas/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Trasplante de Neoplasias , Neoplasias Experimentales , Polisacáridos/química , Polisacáridos/inmunología , Unión Proteica , Transducción de Señal/efectos de los fármacosRESUMEN
We earlier reported the mitogenic and immunostimulatory activities of Rhizoctonia bataticola lectin (RBL), purified from phytopathogenic fungus R. bataticola in human PBMC. The lectin demonstrates specificity towards glycoproteins containing complex N-glycans. Since CD45-protein tyrosine phosphatase that abundantly expresses N-glycans is important in T-cell signaling, the study aimed to investigate the involvement of CD45 in the immunomodulatory activities of RBL. Flowcytometry and confocal microscopy studies revealed that RBL exhibited binding to PBMC and colocalized with CD45. The binding was comparable in cells expressing different CD45 isoforms-RA, -RB and -RO. CD45 blocking antibody reduced the binding and proliferation of PBMC induced by RBL. CD45-PTPase inhibitor dephostatin inhibited RBL-induced proliferation, expression of CD25 and pZAP-70. RBL-induced secretion of Th1/Th2 cytokines were significantly inhibited in presence of dephostatin. Also, dephostatin blocked phosphorylation of p38MAPK and STAT-5 that was crucial for the biological functions of RBL. The study demonstrates the involvement of CD45-mediated signaling in RBL-induced PBMC proliferation and Th1/Th2 cytokine secretion through activation of p38MAPK and STAT-5.
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Citocinas/metabolismo , Inmunomodulación , Lectinas/inmunología , Antígenos Comunes de Leucocito/inmunología , Rhizoctonia/inmunología , Células TH1/inmunología , Células Th2/inmunología , Proliferación Celular , Células Cultivadas , Humanos , Leucocitos Mononucleares/inmunología , Fosforilación , Factor de Transcripción STAT5/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Ophthalmic mycoses caused by infectious fungi are being recognized as a serious concern since they lead to total blindness. Cephalosporium is one amongst several opportunistic fungal species implicated in ophthalmic infections leading to mycotic keratitis. A mitogenic lectin has been purified from the mycelia of fungus Cephalosporium, isolated from the corneal smears of a keratitis patient. Cephalosporium lectin (CSL) is a tetramer with subunit mass of 14 kDa, agglutinates human A, B, and O erythrocytes, and exhibits high affinity for mucin compared to fetuin and asialofetuin but does not bind to simple sugars indicating its complex sugar specificity. CSL showed strong binding to normal human peripheral blood mononuclear cells (PBMCs) to elicit mitogenic activity. The sugar specificity of the lectin and its interaction with PBMCs to exhibit mitogenic effect indicate its possible role in adhesion and infection process of Cephalosporium.
RESUMEN
BACKGROUND: Rhizoctonia bataticola lectin (RBL), purified from phytopathogenic fungus Rhizoctonia bataticola is highly mitogenic towards human peripheral blood mononuclear cells (PBMC). The lectin has sugar specificity towards N-glycans and binds to glycoproteins containing complex N-glycans (Nagre et al., Glycoconj J. 2010). In this study, we investigated the role of Mitogen Activated Protein Kinase (MAPK) and Signal Transducers and Activators of Transcription (STAT)-5 signaling in RBL-induced proliferation and production of Th1/Th2 cytokines. METHODS: Human PBMC were stimulated with RBL and proliferation was determined by tritiated thymidine incorporation assay, cytokine profiles by ELISA and activation of MAPK and STAT-5 by western blotting. RBL binding was monitored by immunofluorescence staining. Expression of IL-2Rα (CD25) was measured by flow cytometry. RESULTS: The binding and mitogenic activities of RBL were inhibited by glycoproteins- mucin, asialofetuin and fetuin. RBL stimulated expression of IL-2Rα and production of Th1/Th2 cytokines- IL-2, IFN-γ, IL-4 and IL-10. RBL-induced phosphorylation of ERK1/2 and p38 MAPK was detected at 1h and 3h respectively. Significant phosphorylation of STAT-5 (tyr(694)) was observed at 12h. Pharmacological inhibitors of p38 MAPK (SB203580) and JAK/STAT (AG490) but not ERK (PD98059) abrogated proliferation. RBL-induced expression of IL-2Rα and secretion of cytokines were drastically inhibited by SB203580 and AG490. CONCLUSIONS: RBL-induced proliferation and production of Th1/Th2 cytokines are mediated via p38 MAPK and STAT-5 signaling. GENERAL SIGNIFICANCE: RBL, a lectin with complex sugar specificity, is strongly mitogenic to human PBMC and stimulates the production of Th1 and Th2 cytokines. The results identified the signaling mechanism underlying the immunostimulatory activity of RBL.
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Citocinas/metabolismo , Proteínas Fúngicas/farmacología , Lectinas/farmacología , Leucocitos Mononucleares/efectos de los fármacos , Rhizoctonia/metabolismo , Transducción de Señal/efectos de los fármacos , Western Blotting , Proliferación Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Proteínas Fúngicas/metabolismo , Humanos , Imidazoles/farmacología , Interferón gamma/metabolismo , Interleucina-10/metabolismo , Interleucina-2/metabolismo , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Interleucina-4/metabolismo , Lectinas/metabolismo , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/metabolismo , Mitógenos/metabolismo , Mitógenos/farmacología , Unión Proteica , Piridinas/farmacología , Factor de Transcripción STAT5/metabolismo , Tirfostinos/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
A lectin with strong mitogenic activity towards human peripheral blood mononuclear cells (PBMCs) and cytotoxic effect on human ovarian cancer cells has been purified from the mycelium of a phytopathogenic fungus, Rhizoctonia bataticola, using ion exchange chromatography and affinity chromatography on asialofetuin-Sepharose. The lectin, termed RBL, is a tetramer of 11-kDa subunits and has unique amino acid sequence at its blocked N-terminus. The purified RBL was blood group nonspecific and its hemagglutination activity was inhibited by mucin (porcine stomach), fetuin (fetal calf serum) and asialofetuin. Glycan array analysis revealed high affinity binding of RBL towards N-glycans and also the glycoproteins containing complex N-glycan chains. Interestingly, the lectin showed high affinity for glycans which are part of ovarian cancer marker CA125, a high molecular weight mucin containing high mannose and complex bisecting type N-linked glycans as well core 1 and 2 type O-glycans. RBL bound to human PBMCs eliciting strong mitogenic response, which could be blocked by mucin, fetuin and asialofetuin demonstrating the carbohydrate-mediated interaction with the cells. Analysis of the kinetics of binding of RBL to PBMCs revealed a delayed mitogenic response indicating a different signaling pathway compared to phytohemagglutinin-L. RBL had a significant cytotoxic effect on human ovarian cancer cell line, PA-1.
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Supervivencia Celular/efectos de los fármacos , Hongos/química , Lectinas/metabolismo , Lectinas/farmacología , Micelio/química , Animales , Línea Celular , Línea Celular Tumoral , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Femenino , Citometría de Flujo , Glicoproteínas/metabolismo , Pruebas de Hemaglutinación , Humanos , Concentración de Iones de Hidrógeno , Lectinas/química , Lectinas/aislamiento & purificación , Peso Molecular , Neoplasias Ováricas/metabolismo , Polisacáridos/metabolismo , Unión Proteica , ConejosRESUMEN
A mannose-binding lectin (RVL) was purified from the tubers of Remusatia vivipara, a monocot plant by single-step affinity chromatography on asialofetuin-Sepharose 4B. RVL agglutinated only rabbit erythrocytes and was inhibited by mucin, asialomucin, asialofetuin and thyroglobulin. Lectin activity was stable up to 80 degrees C and under wide range of pH (2.0-9.3). SDS-PAGE and gel filtration results showed the lectin is a homotetramer of Mr 49.5 kDa, but MALDI analysis showed two distinct peaks corresponding to subunit mass of 12 kDa and 12.7 kDa. Also the N-terminal sequencing gave two different sequences indicating presence of two polypeptide chains. Cloning of RVL gene indicated posttranslational cleavage of RVL precursor into two mature polypeptides of 116 and 117 amino-acid residues. Dynamic light scattering (DLS) and gel filtration studies together confirmed the homogeneity of the purified lectin and supported RVL as a dimer with Mr 49.5 kDa derived from single polypeptide precursor of 233 amino acids. Purified RVL exerts potent nematicidal activity on Meloidogyne incognita, a root knot nematode. Fluorescent confocal microscopic studies demonstrated the binding of RVL to specific regions of the alimentary-tract and exhibited a potent toxic effect on M. incognita. RVL-mucin complex failed to interact with the gut confirming the receptor mediated lectin interaction. Very high mortality (88%) rate was observed at lectin concentration as low as 30 microg/ml, suggesting its potential application in the development of nematode resistant transgenic-crops.