RESUMEN
Immunoglobulin A (IgA) is the main antibody isotype secreted into the intestinal lumen. IgA plays a critical role in the defence against pathogens and in the maintenance of intestinal homeostasis. However, how secreted IgA regulates gut microbiota is not completely understood. In this study, we isolated monoclonal IgA antibodies from the small intestine of healthy mouse. As a candidate for an efficient gut microbiota modulator, we selected a W27 IgA, which binds to multiple bacteria, but not beneficial ones such as Lactobacillus casei. W27 could suppress the cell growth of Escherichia coli but not L. casei in vitro, indicating an ability to improve the intestinal environment. Indeed W27 oral treatment could modulate gut microbiota composition and have a therapeutic effect on both lymphoproliferative disease and colitis models in mice. Thus, W27 IgA oral treatment is a potential remedy for inflammatory bowel disease, acting through restoration of host-microbial symbiosis.
Asunto(s)
Colitis/prevención & control , Microbioma Gastrointestinal/inmunología , Inmunoglobulina A Secretora/inmunología , Enfermedades Inflamatorias del Intestino/prevención & control , Animales , Anticuerpos Monoclonales , Colitis/inmunología , Colitis/microbiología , Escherichia coli/crecimiento & desarrollo , Escherichia coli/inmunología , Femenino , Homeostasis , Enfermedades Inflamatorias del Intestino/inmunología , Enfermedades Inflamatorias del Intestino/microbiología , Intestino Delgado/inmunología , Intestino Delgado/microbiología , Intestinos/inmunología , Intestinos/microbiología , Masculino , Ratones , Ratones Endogámicos BALB C , SimbiosisRESUMEN
Neuropeptides were used in experiments to assess their effects on planarian cells. Intact and decapitated planarians exposed to 10(-6) M neuropeptides for two days were examined electron microscopically and quantitative changes in the nuclear pores and chromatoid bodies in various types of cells were ascertained. The data clearly indicated the diversity of effects produced in planarian cells by neuropeptide treatments. The number of nuclear pores increased considerably in each cell type treated with neuropeptides. In particular, the effects of neuropeptides were strongest in differentiating cells which were forming the regeneration blastema. Neuropeptide-treated cells also experienced a dramatic increase in the number of chromatoid bodies. The results obtained in this study suggest that synthesis of RNAs leading to increases in the numbers of nuclear pores and chromatoid bodies is facilitated in neuropeptide-treated cells which are undergoing cell differentiation. The different mechanisms of the effects induced in undifferentiated cells by neuropeptides are discussed.
Asunto(s)
Diferenciación Celular/fisiología , Neuropéptidos/farmacología , Poro Nuclear/efectos de los fármacos , Planarias/fisiología , Animales , Microscopía Electrónica , Sistemas Neurosecretores/citología , Sistemas Neurosecretores/ultraestructura , Membrana Nuclear/ultraestructura , Poro Nuclear/ultraestructura , Especificidad de Órganos , Regeneración/fisiologíaRESUMEN
The calcareous sponge Leucosolenia laxa releases free-swimming hollow larvae called coeloblastulae that are the characteristic larvae of the subclass Calcinea. Although the coeloblastula is a major type of sponge larva, our knowledge about its development is scanty. Detailed electron microscopic studies on the metamorphosis of the coeloblastula revealed that the larva consists of four types of cells: flagellated cells, bottle cells, vesicular cells, and free cells in a central cavity. The flagellated cells, the principal cell type of the larva, are arranged in a pseudostratified layer around a large central cavity. The larval flagellated cells characteristically have glutinous granules that are used as internal markers during metamorphosis. After a free-swimming period the larva settles on the substratum, and settlement apparently triggers the initiation of metamorphosis. The larval flagellated cells soon lose their flagellum and begin the process of dedifferentiation. Then the larva becomes a mass of dedifferentiated cells in which many autophagosomes are found. Within 18 h after settlement, the cells at the surface of the cell mass differentiate to pinacocytes. The cells beneath the pinacoderm differentiate to scleroblasts that form triradiate spicules. Finally, the cells of the inner cell mass differentiate to choanocytes and are arranged in a choanoderm that surrounds a newly formed large gastral cavity. We found glutinous granules in these three principal cell types of juvenile sponges, thus indicating the multipotency of the flagellated cells of the coeloblastula.
Asunto(s)
Larva/crecimiento & desarrollo , Larva/ultraestructura , Metamorfosis Biológica , Poríferos/crecimiento & desarrollo , Animales , Diferenciación Celular , Núcleo Celular/ultraestructura , Cilios/ultraestructura , Citoplasma/ultraestructura , Gránulos Citoplasmáticos/ultraestructura , Aparato de Golgi/ultraestructura , Microscopía Electrónica , Vacuolas/ultraestructuraRESUMEN
Tyrosinase inhibitory activity of flavonols, galangin, kaempferol and quercetin, was found to come from their ability to chelate copper in the enzyme. In contrast, the corresponding flavones, chrysin. apigenin and luteolin, did not chelate copper in the enzyme. The chelation mechanism seems to be specific to flavonols as long as the 3-hydroxyl group is free. Interestingly, flavonols affect the enzyme activity in different ways. For example, quercetin behaves as a cofactor and does not inhibit monophenolase activity. On the other hand, galangin inhibits monophenolase activity and does not act as a cofactor. Kaempferol neither acts as a cofactor nor inhibits monophenolase activity. However, these three flavonols are common to inhibit diphenolase activity by chelating copper in the enzyme.
Asunto(s)
Flavanonas , Flavonoides/farmacología , Quempferoles , Monofenol Monooxigenasa/antagonistas & inhibidores , Quercetina/análogos & derivados , Arnica/química , Sitios de Unión , Catecol Oxidasa/antagonistas & inhibidores , Catecol Oxidasa/química , Quelantes/metabolismo , Quelantes/farmacología , Cobre/metabolismo , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Flavonas , Flavonoides/metabolismo , Flavonoles , Proteínas Fúngicas/antagonistas & inhibidores , Proteínas Fúngicas/química , Concentración 50 Inhibidora , Cinética , Levodopa/metabolismo , Monofenol Monooxigenasa/química , Oxidación-Reducción , Extractos Vegetales/química , Plantas Medicinales , Quercetina/metabolismo , Quercetina/farmacología , Espectrofotometría , Relación Estructura-ActividadRESUMEN
Tyrosinase inhibitory and antioxidant activity of gallic acid and its series of alkyl chain esters were investigated. All inhibited the oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA) catalyzed by mushroom tyrosinase. However, gallic acid and its short alkyl chain esters were oxidized as substrates yielding the colored oxidation products. In contrast, the long alkyl chain esters inhibited the enzyme activity without being oxidized. This indicates that the carbon chain length is associated with their tyrosinase inhibitory activity, presumably by interacting with the hydrophobic protein pocket in the enzyme. On the other hand, the esters, regardless their carbon chain length, showed potent scavenging activity on the autoxidation of linoleic acid and 1,1-diphenyl-2-p-picryhydrazyl (DPPH) radical, suggesting that the alkyl chain length is not related to the activity. The effects of side-chain length of gallates in relation to their antibrowning activity are studied.
Asunto(s)
Antioxidantes/síntesis química , Ácido Gálico/análogos & derivados , Ácido Gálico/síntesis química , Monofenol Monooxigenasa/antagonistas & inhibidores , Agaricales/enzimología , Antioxidantes/farmacología , Diseño de Fármacos , Conservación de Alimentos , Ácido Gálico/farmacología , Cinética , Oxidación-Reducción , Relación Estructura-ActividadRESUMEN
A common flavonol, kaempferol, isolated from the fresh flower petals of Crocus sativus L. (Iridaceae) was found to inhibit the oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA) catalyzed by mushroom tyrosinase with an ID(50) of 67 microgram/mL (0.23 mM). Interestingly, its 3-O-glycoside derivatives did not inhibit this oxidation. The inhibition kinetics analyzed by a Lineweaver-Burk plot found kaempferol to be a competitive inhibitor, and this inhibitory activity presumably comes from its ability to chelate copper in the enzyme. This copper chelation mechanism can be applicable for all of the flavonols as long as their 3-hydroxyl group is free. However, quercetin, kaempferol, and galangin each affect the oxidation of L-tyrosine in somewhat different ways.
Asunto(s)
Flavonoides/química , Quempferoles , Liliaceae , Monofenol Monooxigenasa/antagonistas & inhibidores , Quercetina/análogos & derivados , Flavonoides/aislamiento & purificación , Humanos , Melaninas/antagonistas & inhibidores , Extractos Vegetales/química , Quercetina/química , Quercetina/aislamiento & purificaciónRESUMEN
A series of alpha,beta-unsaturated aldehydes, otherwise known as (2E)-alkenals, characterized from the olive Olea europaea L. (Oleaceae) oil flavor was found to inhibit the oxidation of L-3, 4-dihydroxyphenylalanine (L-DOPA) catalyzed by mushroom tyrosinase, and the inhibition kinetics analyzed by a Lineweaver-Burk plot found that they are noncompetitive inhibitors. The inhibition mechanism presumably comes from their ability to form a Schiff base with a primary amino group in the enzyme. In addition, the hydrophobic alkyl chain length from the hydrophilic enal group seems to relate to their affinity to the enzyme, and this results in their inhibitory potency.
Asunto(s)
Aromatizantes/farmacología , Monofenol Monooxigenasa/antagonistas & inhibidores , Aceites de Plantas , Cinética , Modelos Químicos , Aceite de Oliva , Relación Estructura-ActividadRESUMEN
The cellular form of prion protein is a precursor of the infectious isoform, which causes fatal neurodegenerative diseases through intermolecular association. One of the characteristics of the prion protein is a high affinity for Cu(II) ions. The site of Cu(II) binding is considered to be the N-terminal region, where the octapeptide sequence PHGGGWGQ repeats 4 times in tandem. We have examined the Cu(II) binding mode of the octapeptide motif and its pH dependence by Raman and absorption spectroscopy. At neutral and basic pH, the single octapeptide PHGGGWGQ forms a 1:1 complex with Cu(II) by coordinating via the imidazole N pi atom of histidine together with two deprotonated main-chain amide nitrogens in the triglycine segment. A similar 1:1 complex is formed by each octapeptide unit in (PHGGGWGQ)2 and (PHGGGWGQ)4. Under weakly acidic conditions (pH approximately 6), however, the Cu(II)-amide- linkages are broken and the metal binding site of histidine switches from N pi to N tau to share a Cu(II) ion between two histidine residues of different peptide chains. The drastic change of the Cu(II) binding mode on going from neutral to weakly acidic conditions suggests that the micro-environmental pH in the brain cell regulates the Cu(II) affinity of the prion protein, which is supposed to undergo pH changes in the pathway from the cell surface to endosomes. The intermolecular His(N tau)-Cu(II)-His(N tau) bridge may be related to the aggregation of prion protein in the pathogenic form.
Asunto(s)
Cobre/química , Cobre/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Priones/química , Priones/metabolismo , Animales , Óxido de Deuterio , Humanos , Concentración de Iones de Hidrógeno , Modelos Moleculares , Unión Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Secuencias Repetitivas de Aminoácido , Espectrofotometría , Espectrometría Raman , AguaRESUMEN
By bioassay-guided fractionation using mushroom tyrosinase (EC 1.14.18.1), 2-hydroxy-4-methoxybenzaldehyde was characterized as the principal tyrosinase inhibitor from three East African medicinal plants, the root of Mondia whitei (Hook) Skeels (Asclepiaceae), the root of Rhus vulgaris Meikle (Anacardiaceae), and the bark of Sclerocarya caffra Sond (Anacardiaceae). It inhibited the oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA) by mushroom tyrosinase with an ID50 of 4.3 micrograms/ml (0.03 mM). The inhibition kinetics analyzed by a Lineweaver-Burk plot found this simple benzaldehyde derivative to be a mixed type inhibitor for this oxidation and affects on the enzyme in several ways. Based on finding this potent tyrosinase inhibitor, various related analogues were also tested in order to gain new insights into their inhibitory functions on a molecular basis.
Asunto(s)
Benzaldehídos/farmacología , Inhibidores Enzimáticos/farmacología , Monofenol Monooxigenasa/antagonistas & inhibidores , Plantas Medicinales/química , Animales , Antineoplásicos Fitogénicos/aislamiento & purificación , Antineoplásicos Fitogénicos/farmacología , Benzaldehídos/aislamiento & purificación , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/aislamiento & purificación , Humanos , Cinética , Melanoma/patología , Ratones , Células Tumorales CultivadasRESUMEN
The regeneration blastema consists of three cell types, undifferentiated cells, regenerative cells, and differentiated cells all of which can be identified by their ultrastructural characteristics. Quantitative changes in the numbers of these cells within the blastema offer important clues about the detailed process of regeneration. When decapitated worms were allowed to regenerate in neuropeptide-containing tap water, the undifferentiated cells accumulated rapidly and initiated blastema formation. These cells were then replaced with regenerative cells, and subsequently with differentiated cells. In the non-treated regenerating worms, the proportion of undifferentiated cells was much lower than in the neuropeptide-treated ones. The results of this study indicate that neuropeptides have a remarkable effect on the early stage of regeneration in planarians. Immunoelectron microscopy using a gold-conjugated anti-neuropeptide antiserum revealed the presence of neuropeptides in some kinds of parenchymal cells in the post-blastemal area.
Asunto(s)
Neuropéptidos/farmacología , Planarias/efectos de los fármacos , Planarias/crecimiento & desarrollo , Animales , Diferenciación Celular/efectos de los fármacos , Inmunohistoquímica , Microscopía Inmunoelectrónica , Neuropéptidos/metabolismo , Planarias/ultraestructura , Regeneración/efectos de los fármacosRESUMEN
The Caenorhabditis elegans unc-18-encoded protein (UNC-18) is implicated in the processes of vesicle targeting, docking, and/or fusion. To further characterize the properties of this important neural protein, we expressed it at a high level in Spodoptera frugiperda Sf21 cells using a baculovirus expressing system. A cDNA containing the coding sequence for UNC-18 was inserted into the transfer vector pBlueBac to yield the recombinant virus pAcNPV/unc-18. At maximal expression, the recombinant virus produces a protein of 67 kDa, which constitutes about one-third of total cell protein. The UNC-18 protein was highly purified and its biochemical and functional properties were assessed. The protein is globular with an isoelectric point of 6.95. Circular dichroism spectroscopy indicated that the alpha-helix and beta-sheet account for 10.0 and 59.0%, respectively. Immunolabeling the Sf21 cells expressing UNC-18 showed that the expressed UNC-18 is predominantly localized in the cytoplasm as a soluble monomer. The protein is phosphorylated by protein kinase C and binds to the recombinant C. elegans syntaxin in vitro. These findings suggest that in vesicle traffic UNC-18 is a regulator factor associated with the plasma membrane through syntaxin, although intrinsically cytoplasmic.
Asunto(s)
Baculoviridae/genética , Proteínas de Caenorhabditis elegans , Proteínas Portadoras , Clonación Molecular/métodos , Vectores Genéticos , Proteínas del Helminto/genética , Fosfoproteínas , Proteínas de Transporte Vesicular , Animales , Caenorhabditis elegans , Proteínas del Helminto/biosíntesis , Proteínas del Helminto/aislamiento & purificación , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Fosforilación , Estructura Secundaria de Proteína , Proteínas Qa-SNARE , Proteínas Recombinantes/biosíntesis , Análisis de Secuencia , Spodoptera , Fracciones Subcelulares/químicaRESUMEN
Prion diseases share a common feature in that the normal cellular prion protein (PrP(C)) converts to a protease-resistant isoform PrP(Sc). The alpha-helix-rich C-terminal half of PrP(C) is partly converted into beta-sheet in PrP(Sc). We have examined by Raman spectroscopy the structure of an octapeptide PHGGGWGQ that appears in the N-terminal region of PrP(C) and a longer peptide containing the octapeptide region. The peptides do not assume any regular structure without divalent metal ions, whereas Cu(II) binding to the HGGG segment induces formation of alpha-helical structure on the C-terminal side of the peptide chain. The N-terminal octapeptide of prion protein may be a novel structural motif that acts as a promoter of alpha-helix formation.
Asunto(s)
Cobre/metabolismo , Fragmentos de Péptidos/química , Proteínas PrPC/química , Estructura Secundaria de Proteína , Secuencia de Aminoácidos , Sitios de Unión , Glicina/análisis , Humanos , Datos de Secuencia Molecular , Fragmentos de Péptidos/metabolismo , Proteínas PrPC/metabolismo , Espectrometría RamanRESUMEN
The free-swimming parenchymella larvae of Haliclona permollis have a surface of flagellated cells that function in locomotion. These flagellated cells disappear from the larval surface soon after larval settlement, but the debate about their fate during metamorphosis has not been resolved. An improved method for electron microscopy enabled us to visualize minute ellipsoid granules characteristic of the larval flagellated cells of this sponge and to use these granules as natural markers to follow the fate of flagellated cells in metamorphs. On metamorphosis, the axonemes of the flagellated cells are withdrawn into the cell body and persist for some time, thus serving as a second natural marker for flagellated cells in postlarval forms. Within 12 h after settlement, the metamorphs have both markers in amoeboid cells derived from flagellated cells. The minute ellipsoid granules are found in the amoeboid cells of the metamorphs 24 h after settlement, and in the choanocytes of the juveniles 36 h after settlement. Therefore, it is proposed that the choanocytes derive from the larval flagellated cells by way of an amoeboid cell stage. These results suggest that the flagellated cells of parenchymella larvae participate in the formation of juveniles.
RESUMEN
Bioassay-guided fractionation monitored by mushroom tyrosinase (EC 1.14.18.1) activity, afforded six inhibitors from three Bolivian medicinal plants, Buddleia coriacea, Gnaphalium cheiranthifolium, and Scheelea princeps. These inhibitors, which are all known phenolic compounds, inhibited the oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA) mediated by a mushroom tyrosinase.
Asunto(s)
Monofenol Monooxigenasa/antagonistas & inhibidores , Plantas Medicinales/química , Basidiomycota/enzimología , BoliviaRESUMEN
Mutations in the Caenorhabditis elegans unc-41 gene result in an allele-dependent elevation of acetylcholine content. Eight recessive alleles (cn252, e268, e399, e650, e1175, e1199, e1294, and e870) lead to phenotypes including uncoordinated locomotion, slow growth, a small mature body, and resistance to the acetylcholinesterase inhibitors as well as the elevation of acetylcholine content. The remaining two alleles, e554 and e1162, exhibit normal acetylcholine levels but display the short-body phenotype in a semidominant way. To determine the localization of the elevated acetylcholine content, a method for the isolation of synaptic vesicles from C. elegans was established. The elevation of acetylcholine content in the unc-41 mutants is accompanied by the accumulation of synaptic vesicles. We propose that at least one function of the unc-41 gene relates to the release of neurotransmitters.
Asunto(s)
Acetilcolina/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Mutación , Acetilcolinesterasa/metabolismo , Alelos , Animales , Caenorhabditis elegans/crecimiento & desarrollo , Colina O-Acetiltransferasa/metabolismo , Cruzamientos Genéticos , Trastornos del Desarrollo Sexual , Prueba de Complementación Genética , Masculino , Actividad Motora , Supresión Genética , Vesículas Sinápticas/metabolismoRESUMEN
Anacardic acids, 2-methylcardols, and cardols isolated from various parts of the cashew [Anacardium occidentale] (Anacardiaceae) fruit have been found to exhibit tyrosinase inhibitory activity. Kinetic studies with the two principal active compounds, 6-[8(Z),11(Z),14-pentadecatrienyl]salicylic acid and 5-[8(Z),11(Z),14-pentadecatrienyl]resorcinol, have indicated that both of these phenolic compounds exhibit characteristic competitive inhibition of the oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA) by mushroom tyrosinase.
Asunto(s)
Ácidos Anacárdicos , Monofenol Monooxigenasa/antagonistas & inhibidores , Nueces/química , Resorcinoles/aislamiento & purificación , Salicilatos/aislamiento & purificación , Basidiomycota/enzimología , Cinética , Levodopa/metabolismo , Monofenol Monooxigenasa/metabolismo , Oxidación-Reducción , Resorcinoles/farmacología , Salicilatos/farmacología , Relación Estructura-ActividadRESUMEN
The C. elegans unc-18 gene is required to maintain normal acetylcholine levels. We determined the complete structure of an unc-18 cDNA that encodes a protein of 591 highly charged and hydrophilic amino acids. The protein shows sequence similarity with elements of the secretory pathway in the yeast S. cerevisiae. Antibodies raised against a portion of the unc-18-encoded protein (UNC-18) detected a 68 kd soluble antigen on immunoblots and intensely stained all vertical cord motor neurons in situ. These findings suggest that UNC-18 participates in the axonal transport system and influences the acetylcholine flow in motor neurons.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans/genética , Proteínas Portadoras , Proteínas del Helminto/biosíntesis , Proteínas del Helminto/genética , Neuronas Motoras/metabolismo , Fosfoproteínas , Proteínas de Transporte Vesicular , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Caenorhabditis elegans/citología , Caenorhabditis elegans/metabolismo , Cartilla de ADN , ADN Complementario/análisis , Expresión Génica , Proteínas del Helminto/química , Immunoblotting , Larva , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/métodos , Mapeo Restrictivo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de AminoácidoAsunto(s)
Genes ras , Neoplasias Pulmonares/secundario , Animales , Southern Blotting , Línea Celular , Línea Celular Transformada , Exones , Genes myc , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Ratones , Ratones Endogámicos BALB C , Metástasis de la Neoplasia , TransfecciónRESUMEN
OBJECTIVE: Sleep apnea is often caused by obstruction of the pharyngeal airway. The goal of this study was to use ultrafast MR imaging to examine the pharyngeal airway in patients with sleep apnea and to evaluate the usefulness of this technique for localizing the site of obstruction. SUBJECTS AND METHODS: Fifteen patients with sleep apnea and five healthy volunteers underwent ultrafast MR imaging while awake and during sleep induced with hydroxyzine hydrochloride. Sequential midline sagittal images of the pharynx were obtained and displayed in the cine mode. RESULTS: Patients with sleep apnea were found to have sites of pharyngeal abnormality that were not present in healthy volunteers. Nine sites of narrowing in seven patients (47%) were detected with the patient awake; 21 sites of obstruction in 13 patients (87%) were diagnosed with the patient asleep. Six patients showed only one obstruction, and seven had several obstructions: five had obstructions at the velum palatinum and at the oropharynx; one had obstructions at the velum palatinum, oropharynx, and hypopharynx; one had obstructions at the velum palatinum and the hypopharynx. The sites of narrowing during wakefulness and the sites of obstruction during sleep were the same in only four (31%) of the patients with pharyngeal airway obstruction. CONCLUSION: Ultrafast MR imaging is useful for localizing the sites of pharyngeal airway obstruction in patients with sleep apnea.