RESUMEN
To search for low-energy resonant structures in isospin T=3/2 three-body systems, we have performed the experiments ^{3}H(t,^{3}He)3n and ^{3}He(^{3}He,t)3p at intermediate energies. For the 3n experiment, we have newly developed a thick Ti-^{3}H target that has the largest tritium thickness among targets of this type ever made. The 3n experiment for the first time covered the momentum-transfer region as low as 15 MeV/c, which provides ideal conditions for producing fragile systems. However, in the excitation-energy spectra we obtained, we did not observe any distinct peak structures. This is in sharp contrast to tetraneutron spectra. The distributions of the 3n and 3p spectra are found to be similar, except for the displacement in energy due to Coulomb repulsion. Comparisons with theoretical calculations suggest that three-body correlations exist in the 3n and 3p systems, although not enough to produce a resonant peak.
RESUMEN
We performed the first direct mass measurements of neutron-rich scandium, titanium, and vanadium isotopes around the neutron number 40 at the RIKEN RI Beam Factory using the time-of-flight magnetic-rigidity technique. The atomic mass excesses of ^{58-60}Sc, ^{60-62}Ti, and ^{62-64}V were measured for the first time. The experimental results show that the two-neutron separation energies in the vicinity of ^{62}Ti increase compared to neighboring nuclei. This shows that the masses of Ti isotopes near N=40 are affected by the Jahn-Teller effect. Therefore, a development of Jahn-Teller stabilization appears below the Cr isotopes, and the systematics in Sc, Ti, and V isotopes suggest that ^{62}Ti is located close to the peak of the Jahn-Teller effect.
RESUMEN
We perform the first direct mass measurements of neutron-rich calcium isotopes beyond neutron number 34 at the RIKEN Radioactive Isotope Beam Factory by using the time-of-flight magnetic-rigidity technique. The atomic mass excesses of ^{55-57}Ca are determined for the first time to be -18650(160), -13510(250), and -7370(990) keV, respectively. We examine the emergence of neutron magicity at N=34 based on the new atomic masses. The new masses provide experimental evidence for the appearance of a sizable energy gap between the neutron 2p_{1/2} and 1f_{5/2} orbitals in ^{54}Ca, comparable to the gap between the neutron 2p_{3/2} and 2p_{1/2} orbitals in ^{52}Ca. For the ^{56}Ca nucleus, an open-shell property in neutrons is suggested.
RESUMEN
The cross sections of the ^{7}Be(n,α)^{4}He reaction for p-wave neutrons were experimentally determined at E_{c.m.}=0.20-0.81 MeV slightly above the big bang nucleosynthesis (BBN) energy window for the first time on the basis of the detailed balance principle by measuring the time-reverse reaction. The obtained cross sections are much larger than the cross sections for s-wave neutrons inferred from the recent measurement at the n_TOF facility in CERN, but significantly smaller than the theoretical estimation widely used in the BBN calculations. The present results suggest the ^{7}Be(n,α)^{4}He reaction rate is not large enough to solve the cosmological lithium problem, and this conclusion agrees with the recent result from the direct measurement of the s-wave cross sections using a low-energy neutron beam and the evaluated nuclear data library ENDF/B-VII.1.
RESUMEN
BACKGROUND: Neuroimmunological disorders are involved in the pathogenesis of atopic dermatitis (AD), partly through enhanced sensory nerve-skin mast cell interaction. Cell adhesion molecule 1 (CADM1) is a mast-cell adhesion molecule that mediates the adhesion to, and communication with, sympathetic nerves. OBJECTIVES: To investigate the role of mast cell CADM1 in the pathogenesis of AD, CADM1 expression levels by comparing between lesional and nonlesional skin mast cells of an AD mouse model, which was developed by repeated application of trinitrochlorobenzene, and to examine, in cocultures, how the alterations in CADM1 detected in lesional mast cells might affect the sensory nerve-mast cell interaction. METHODS: AD-like lesional and nonlesional skin mast cells were collected separately by laser capture microdissection. CADM1 expression was examined by reverse transcription-polymerase chain reaction and CADM1 immunohistochemistry. In cocultures, adhesion between dorsal root ganglion (DRG) neurites and IC2 mast cells was analysed by loading a femtosecond laser-induced impulsive force on neurite-attendant IC2 cells, while cellular communication was monitored as the IC2 cellular response ([Ca(2+)]i increase) after nerve-specific stimulant-induced DRG activation. RESULTS: AD-like lesional mast cells expressed three-fold more CADM1 transcripts than nonlesional cells. This was supported at the protein level, shown by immunohistochemistry. In coculture, CADM1 overexpression in IC2 cells strengthened DRG neurite-IC2 cell adhesion and doubled the population of IC2 cells responding to DRG activation. A function-blocking anti-CADM1 antibody abolished these effects in a dose-dependent manner. CONCLUSIONS: Increased expression of CADM1 in mast cells appeared to be a cause of enhanced sensory nerve-mast cell interaction in a hapten-induced mouse model of AD.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Dermatitis Atópica/metabolismo , Inmunoglobulinas/metabolismo , Mastocitos/metabolismo , Células Receptoras Sensoriales/fisiología , Animales , Adhesión Celular , Molécula 1 de Adhesión Celular , Moléculas de Adhesión Celular/fisiología , Comunicación Celular/fisiología , Células Cultivadas , Dermatitis Atópica/inducido químicamente , Pabellón Auricular , Ganglios Espinales/fisiología , Haptenos/toxicidad , Inmunoglobulinas/fisiología , Ratones , Ratones Endogámicos BALB C , Neuritas/fisiología , Cloruro de Picrilo/toxicidad , Venenos de Escorpión/farmacologíaRESUMEN
OBJECTIVE AND DESIGN: We have studied the role of phosphatidylinositol 4-kinase IIalpha (PI4KIIalpha) in activation of rat basophilic leukemia (RBL-2H3) cells. MATERIALS AND METHODS: Antigen-mediated intracellular Ca(2+) concentration ([Ca(2+)](i)) increase and beta-hexosaminidase secretion were measured using RBL-2H3 cells stably expressing PI4KIIalpha-yellow fluorescent protein (YFP) or its kinase-deficient mutant PI4KIIalpha (K151A)-YFP. RESULTS: Neither PI4KIIalpha-YFP nor PI4KIIalpha (K151A)-YFP were distributed on the plasma membranes but on the exocytotic vesicles. The RBL-2H3 cells stably expressing PI4KIIalpha-YFP showed significantly enhanced beta-hexosaminidase secretion but not an increase in [Ca(2+)](i) after antigen stimulation. The cells with PI4KIIalpha (K151A)-YFP showed no change in the [Ca(2+)](i) increase nor degranulation. The promotion of secretion by PI4KIIalpha-YFP was not observed using co-stimulation with Ca(2+) ionophore and the protein kinase C activator, phorbol myristate acetate. CONCLUSIONS: These results suggest that PI4KIIalpha plays a role in the exocytotic process downstream of Ca(2+) signaling in antigen-mediated mast cell activation.
Asunto(s)
Señalización del Calcio/fisiología , Degranulación de la Célula/fisiología , Leucemia Basofílica Aguda/patología , Leucemia Basofílica Aguda/fisiopatología , Fosfotransferasas (Aceptor de Grupo Alcohol)/fisiología , Animales , Antígenos , Línea Celular Tumoral , Gránulos Citoplasmáticos/fisiología , Exocitosis/fisiología , Regulación Enzimológica de la Expresión Génica , Mastocitos/patología , Mastocitos/fisiología , Antígenos de Histocompatibilidad Menor , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Receptores de IgE/fisiologíaRESUMEN
Increased arterial stiffness is strongly associated with atherosclerosis, while platelet activation is an important trigger of thrombotic events in patients with atherosclerosis. However, little is known about the effect of arterial stiffness on platelet activation. We therefore investigated the association between arterial stiffness and platelet activation in 38 normal volunteers (20 men and 18 women) aged 23-77 years (mean = 49 +/- 15 years). Arterial stiffness was assessed by measuring brachial-ankle pulse wave velocity (ba-PWV) and heart-brachial PWV (hb-PWV). Flow cytometric analyses were performed to evaluate platelet activation by measuring surface expression of P-selectin and platelet-neutrophil complexes (PNC) before and after activation by ADP. We also calculated the difference between basal and stimulated states of P-selectin and PNC to assess platelet activation reserve. PWVs were significantly correlated with age and BP (r = 0.60-0.81). For platelet activation and activation reserve, correlations with age were less strong but remained significant (r = 0.36-0.61), with the exception of P-selectin (not significant, NS), and correlations with SBP were similar (r = 0.35-0.53). A significant correlation was found between PWVs and platelet activation (r = 0.43-0.74). Multiple regression analysis demonstrated significant correlations between platelet activation and reserve and PWVs (coefficient = 2.17-6.59), when both age and BP were adjusted for simultaneously. In conclusion, platelet activation was associated with arterial stiffness, suggesting that arterial stiffness may play an important role in thrombotic events.
Asunto(s)
Arteria Braquial/fisiología , Activación Plaquetaria/fisiología , Flujo Pulsátil/fisiología , Adulto , Anciano , Arteriosclerosis/sangre , Arteriosclerosis/complicaciones , Arteriosclerosis/fisiopatología , Presión Sanguínea , Femenino , Citometría de Flujo , Humanos , Masculino , Persona de Mediana Edad , Recuento de Plaquetas , Pletismografía , Valores de Referencia , Factores de Riesgo , Trombosis/sangre , Trombosis/etiología , Trombosis/fisiopatologíaRESUMEN
A genetic predisposition to the development of neuroleptic malignant syndrome (NMS) has been suggested by clinical studies. Although the molecular basis of NMS is unclear, a dopaminergic blockade mechanism has been considered the main cause. We therefore investigated the association between NMS and three functional polymorphisms of the dopamine D(2) receptor (DRD(2)) gene: TaqI A, -141C Ins/Del, and Ser311Cys. Subjects included 32 Japanese patients, previously diagnosed with NMS, and 132 schizophrenic patients treated with neuroleptics without occurrence of NMS. Polymerase chain reaction and restriction fragment length polymorphism analyses were performed to determine each genotype. We found significant differences in genotypic and allelic frequencies of the -141C Ins/Del polymorphism between patients with and without NMS. The -141C Del allele was significantly more frequent in the NMS group (23.4 vs 11.7%, P=0.026). Similarly, the proportion of -141C Del allele carriers was significantly higher in the NMS group (40.6 vs 20.5%, P=0.022). No significant differences between the two groups were seen for allelic and genotypic frequencies of the TaqI A and Ser311Cys polymorphisms. This result suggests that the -141C Ins/Del polymorphism is likely to predispose toward the development of NMS, probably together with other unidentified factors.
Asunto(s)
Síndrome Neuroléptico Maligno/genética , Polimorfismo Genético/genética , Receptores de Dopamina D2/genética , Sustitución de Aminoácidos , Pueblo Asiatico/genética , Intervalos de Confianza , ADN/sangre , ADN/aislamiento & purificación , Femenino , Frecuencia de los Genes , Tamización de Portadores Genéticos , Genotipo , Humanos , Japón , Leucocitos , Masculino , Persona de Mediana Edad , Oportunidad Relativa , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Eliminación de SecuenciaRESUMEN
Mast cell-neurite interaction serves as a model for neuroimmune interaction. We have shown that neurite-mast cell communication can occur via substance P interacting with neurokinin (NK)-1 receptors on the mucosal mast cell-like cell, the rat basophilic leukemia (RBL) cell. Neurite (murine superior cervical ganglia) and RBL cell [expressing the granule-associated antigen CD63-green fluorescent protein (GFP) conjugate] cocultures were established and stimulated with bradykinin (BK; 10 nM) or scorpion venom (SV; 10 pg/ml), both of which activate only neurites. Cell activation was assessed by confocal imaging of Ca2+ (cells preloaded with fluo 3), and analyses of RBL CD63-GFP+ granule movement were conducted. Neurite activation by BK or SV was followed by RBL Ca2+ mobilization, which was inhibited by an NK-1 receptor antagonist (NK-1 RA). Moreover, membrane ruffling was observed on RBL pseudopodial extensions in contact with the activated neurite, but not on noncontacting pseudopodia. RBL membrane ruffling was inhibited by NK-1 RA, but not NK-2 RA, and was accompanied by a significant increase in granule movement (0.13 +/- 0.04 vs. 0.05 +/- 0.01 microm/s) that was most evident at the point of neurite contact: many of the granules moved toward the plasmalemma. This is the first documentation of such precise (restricted to the membrane's contact site) transfer of information between nerves and mast cells that could allow for very subtle in vivo communication between these two cell types.
Asunto(s)
Comunicación Celular/fisiología , Leucemia Basofílica Aguda/fisiopatología , Mastocitos/fisiología , Ganglio Cervical Superior/fisiopatología , Animales , Membrana Celular/fisiología , Células Cultivadas , Técnicas de Cocultivo , Gránulos Citoplasmáticos/fisiología , Ratones , Ratones Endogámicos CBA , Neuritas/fisiología , RatasRESUMEN
We describe a patient with treatment-resistant schizophrenia who had a duplication in the cytochrome P450IID6 (CYP2D6) gene. This severely ill 71-year-old-woman had responded poorly to several neuroleptics. Molecular genetic study revealed CYP2D6 gene duplication, which results in excessive activity of CYP2D6 that metabolizes various commonly used neuroleptics. The mutation may have contributed to treatment resistance in this case.
Asunto(s)
Citocromo P-450 CYP2D6/genética , Resistencia a Medicamentos/genética , Duplicación de Gen , Esquizofrenia/tratamiento farmacológico , Esquizofrenia/genética , Anciano , Femenino , HumanosRESUMEN
Palmitoyl-CoA (Pal-CoA) lowered the respiratory control ratio (RCR), and induced mitochondrial membrane permeability transition (MPT) and cytochrome c (Cyt. c) release from isolated rat liver mitochondria. L-Carnitine suppressed the Pal-CoA-induced dysfunction, MPT, and Cyt. c release of isolated mitochondria. This suppression was inhibited by cephaloridine, an inhibitor of carnitine uptake into mitochondria. Cyclosporin A (CsA), an inhibitor of MPT, and BSA also suppressed the Pal-CoA-induced MPT. In the presence of inorganic phosphate (P(i)), Ca2+-induced MPT was suppressed by BSA, L-carnitine, and chlorpromazine, an inhibitor of phospholipase A2. In the presence of a low concentration of Ca2+, 3,3',5-triiodothyronine, long chain fatty acids, salicylic acid, and diclofenac induced MPT by a mechanism that was suppressed by BSA, L-carnitine, or chlorpromazine. During the incubation of mitochondria on ice, their respiratory competence decreased; L-carnitine and BSA also prevented this decrease. Mitochondrial depolarization in pheochromocytoma PC12 cells was induced by either serum deprivation or arachidonic acid by a mechanism that was suppressed by acetyl-L-carnitine. These results indicate that some MPTs may be regulated by fatty acid metabolism and that the Pal-CoA-induced MPT plays an important role in the induction of apoptosis.
Asunto(s)
Carnitina/farmacología , Ácidos Grasos/farmacología , Mitocondrias Hepáticas/efectos de los fármacos , Animales , Senescencia Celular/efectos de los fármacos , Cefaloridina/farmacología , Cefalosporinas/farmacología , Clorpromazina/farmacología , Ciclosporina/farmacología , Grupo Citocromo c/metabolismo , Antagonistas de Dopamina/farmacología , Interacciones Farmacológicas , Inhibidores Enzimáticos/farmacología , Mitocondrias Hepáticas/fisiología , Dilatación Mitocondrial/efectos de los fármacos , Células PC12 , Palmitoil Coenzima A/farmacología , Permeabilidad/efectos de los fármacos , Fosforilación , Ratas , Ratas Wistar , Albúmina Sérica Bovina/farmacologíaRESUMEN
Communication between nerves and mast cells is a prototypic demonstration of neuroimmune interaction. We have recently shown that direct nerve-mast cell cross-talk can occur in the absence of an intermediary transducing cell and that the neuropeptide substance P is an important mediator of this communication. Here we study the calcium signals in rat basophilic leukemia cells (RBL-2H3; mucosal-type mast cells) primed with substance P. RBL cells responded only slightly to stimulation with compound 48/80, however they responded to the stimulation when the cells had been primed with substance P (0.5 microM) for one week. The present results provide a foundation to study the neuroimmune cross-talk in a co-culture system.
Asunto(s)
Señalización del Calcio/efectos de los fármacos , Leucemia Basofílica Aguda/metabolismo , Mastocitos/metabolismo , Sustancia P/farmacología , Animales , Colorantes Fluorescentes , Fura-2 , Liberación de Histamina/efectos de los fármacos , Cinética , Ratas , Receptor Cross-Talk/efectos de los fármacos , Espectrometría de Fluorescencia , Estimulación Química , Sustancia P/metabolismo , Células Tumorales Cultivadas , beta-N-Acetilhexosaminidasas/metabolismo , p-Metoxi-N-metilfenetilamina/farmacologíaRESUMEN
We evaluated the activation of mu-calpain in progesterone-activated human sperm. Semen collected from fertile donors with informed consent was liquefied and subjected to percoll gradient centrifugation. After exposure to different concentrations of progesterone, the samples were used for immunostaining, SDS-PAGE and Western blot analysis. An increase of the intracellular free calcium concentration in the sperm following the addition of progesterone was observed using fura-2 AM. Immunostaining using an antibody against active mu-calpain produced 6 distinct staining patterns: (1) the acrosome, (2) an equatorial segment, (3) the whole head, (4) the neck, (5) the neck and tail or (6) unstained sperm. After addition of progesterone, the predominant type changed from the neck type (90%) to the neck and tail type (79%). Western blot analysis using a pro-mu-calpain and a mu-calpain domain III antibody revealed autodigestion of mu-calpain, indicating activation by progesterone. Using calpain-specific inhibitors it was shown that calpain activation contributes to sperm motility as well as to the acrosome reaction. These results suggest the possibility that activation of mu-calpain in human sperm by progesterone plays an important role in fertilization.
Asunto(s)
Calpaína/fisiología , Progesterona/farmacología , Espermatozoides/efectos de los fármacos , Western Blotting , Calcio/metabolismo , Calpaína/metabolismo , Calpaína/farmacología , Activación Enzimática/efectos de los fármacos , Precursores Enzimáticos/metabolismo , Precursores Enzimáticos/farmacología , Precursores Enzimáticos/fisiología , Fertilización , Humanos , Inmunohistoquímica , Masculino , Progesterona/fisiología , Motilidad Espermática/efectos de los fármacos , Espermatozoides/enzimología , Espermatozoides/fisiologíaRESUMEN
CD63 is located on the basophilic granule membranes in resting basophils, mast cells, and platelets, and is also located on the plasma membranes of the cells. We constructed a CD63-GFP (green fluorescent protein) plasmid and introduced it into rat basophilic leukemia (RBL-2H3) cells to observe the movements of CD63 on degranulation. The movements of CD63-GFP were studied in living RBL cells by confocal laser scanning microscopy (CLSM). CD63-GFP, in which GFP was conjugated to the C-terminus of CD63, was located on both the granule membranes and the plasma membranes of RBL cells. The diameter of the fluorescent granules in the cytoplasm varied from 0.5 to 1.5 microm. Before antigen stimulation most granules with CD63-GFP hardly moved in RBL cells. However, after antigen stimulation the plasma membranes ruffled violently and the granules moved dramatically. They reached the plasma membranes in a few minutes and fused with them instantaneously. Analysis of the movement of each granule provided a new insight into the elementary process of degranulation. The velocity of the granule movement toward the plasma membranes on antigen stimulation was calculated to be 0.1+/-0.02 microm/s. This shows that the granules are able to reach the plasma membranes in 2 or 3 min if the diameter of the cells is 20 microm.
Asunto(s)
Antígenos CD/metabolismo , Degranulación de la Célula/fisiología , Gránulos Citoplasmáticos/metabolismo , Proteínas Luminiscentes/metabolismo , Glicoproteínas de Membrana Plaquetaria/metabolismo , Animales , Membrana Celular/metabolismo , Corriente Citoplasmática , Proteínas Fluorescentes Verdes , Inmunización , Leucemia Basofílica Aguda/metabolismo , Leucemia Basofílica Aguda/patología , Microscopía Confocal , Plásmidos/genética , Ratas , Tetraspanina 30 , Células Tumorales Cultivadas , beta-N-Acetilhexosaminidasas/metabolismoAsunto(s)
Fibrilación Atrial/mortalidad , Factor Natriurético Atrial/sangre , Cardiomiopatía Dilatada/mortalidad , Muerte Súbita Cardíaca/epidemiología , Insuficiencia Cardíaca/mortalidad , Adulto , Animales , Fibrilación Atrial/sangre , Fibrilación Atrial/diagnóstico , Cardiomiopatía Dilatada/sangre , Cardiomiopatía Dilatada/diagnóstico , Causas de Muerte , Muerte Súbita Cardíaca/etiología , Supervivencia sin Enfermedad , Femenino , Insuficiencia Cardíaca/sangre , Insuficiencia Cardíaca/diagnóstico , Humanos , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Pronóstico , Factores de RiesgoRESUMEN
Dementia with Lewy bodies (DLB) is the second most frequent degenerative dementia among the elderly, following Alzheimer-type dementia (ATD). An association of DLB with CYP2D6*4, one of the cytochrome P450IID6 (debrisoquine 4-hydroxylase; CYP2D6) gene polymorphisms, was reported previously, but this is controversial. Moreover, these reports have been restricted to Caucasian populations. Therefore, we compared frequencies of CYP2D6*3, *4, and *10 mutant alleles in 17 Japanese DLB patients to those among Alzheimer-type dementia (ATD) patients and healthy controls. Polymerase chain reaction amplification and restriction fragment length polymorphism analyses were used for genotyping. No significant difference of genotype or mutant allele frequencies was detected between DLB, ATD, and healthy controls. The present results do not support the suggestion that the CYP2D6 gene is related to DLB susceptibility, at least in the Japanese population.
Asunto(s)
Citocromo P-450 CYP2D6/genética , Enfermedad por Cuerpos de Lewy/genética , Polimorfismo de Longitud del Fragmento de Restricción , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/epidemiología , Enfermedad de Alzheimer/genética , Técnicas de Cultivo , Femenino , Frecuencia de los Genes/genética , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Japón/epidemiología , Enfermedad por Cuerpos de Lewy/epidemiología , MasculinoRESUMEN
The mitogen-activated protein kinase (MAPK) cascade consists of the MAPK (extracellular signal-regulated kinase 2; ERK2) and its activator, MAPK kinase (MAP/ERK kinase; MEK). However, the mechanisms for activation of ERK2 have not been defined yet in cells. Here, we used fluorescent protein-tagged ERK2 and MEK to examine the localization of ERK2 and MEK in living rat basophilic leukemia (RBL-2H3) cells. ERK2 was mainly in the cytoplasm in resting cells but translocated into the nucleus after the ligation of IgE receptors. The import of ERK2 reached the maximum at 6--7 min, and then the imported ERK2 was exported from the nucleus. MEK mainly resided in the cytoplasm, and no significant MEK translocation was detected statically after ligation of IgE receptors. However, analysis of the dynamics of ERK2 and MEK suggested that both of them rapidly shuttle between the cytoplasm and the nucleus and that MEK regulates the nuclear shuttling of ERK2, whereas MEK remains mainly in the cytoplasm. In addition, the data suggested that the sustained calcium increase was required for the optimal translocation of ERK2 into the nucleus in RBL-2H3 cells. These results gave a new insight of the dynamics of ERK2 and MEK in the nuclear shuttling of RBL-2H3 cells after the ligation of IgE receptors.
Asunto(s)
Antígenos/inmunología , Núcleo Celular/enzimología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/fisiología , Células Tumorales Cultivadas/enzimología , Transporte Activo de Núcleo Celular/genética , Transporte Activo de Núcleo Celular/inmunología , Animales , Proteínas Bacterianas/genética , Calcio/fisiología , Cationes Bivalentes/farmacología , Núcleo Celular/genética , Núcleo Celular/metabolismo , Dinitrofenoles/inmunología , Activación Enzimática/genética , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/genética , Microscopía Confocal , Microscopía Fluorescente , Proteína Quinasa 1 Activada por Mitógenos/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Ratas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Albúmina Sérica Bovina/inmunología , Fracciones Subcelulares/enzimología , Transfección , Células Tumorales Cultivadas/metabolismoRESUMEN
Communication between nerves and mast cells is a prototypic demonstration of neuro-immune interaction. Recently, we used an in vitro co-culture approach comprising cultured murine superior cervical ganglia (SCG) and rat basophilic leukemia (RBL) cells to study this interaction. Previously, we concentrated mainly on the activation signal from neurites to mast cells (RBL). However, it is proposed that mast cell-nerve communication is not a one-sided relationship but a bi-directional one. In the present work, we studied the communication from mast cells to neurites. We observed that binding of anti-IgE receptor antibodies to mast cells increases calcium ion concentration [Ca2+]i in SCG neurites. This indicates that mast cell-nerve communication is bi-directional. Confocal fluorescence microscopic images indicated that [Ca2+]i in neurites increased after an increase of [Ca2+]i in mast cells. The lag-time of neurite activation was several times longer than that of mast cell activation. The correlation coefficient between the lag-times for mast cell and nerve activation was calculated to be 0.81. In addition, the fluorescence images showed that calcium signals in SCG neurites were able to extend to a long distance (100-200 microm) from the site where mast cells (RBL) attached to neurites.
Asunto(s)
Comunicación Celular/fisiología , Mastocitos/fisiología , Neuronas/fisiología , Animales , Animales Recién Nacidos , Calcio/metabolismo , Técnicas de Cocultivo , Técnica del Anticuerpo Fluorescente , Mastocitos/metabolismo , Mastocitos/ultraestructura , Ratones , Ratones Endogámicos CBA , Microscopía Confocal , Neuritas/fisiología , Neuronas/metabolismo , Neuronas/ultraestructura , Receptores de IgE/fisiología , Ganglio Cervical Superior/citologíaRESUMEN
Fluorescence resonance energy transfer (FRET) experiments have been performed to elucidate the structural features of oligonucleotide duplexes containing the pyrimidine(6-4)pyrimidone photoproduct, which is one of the major DNA lesions formed at dipyrimidine sites by UV light. Synthetic 32mer duplexes with and without the (6-4) photoproduct were prepared and fluorescein and tetramethylrhodamine were attached, as a donor and an acceptor, respectively, to the aminohexyl linker at the C5 position of thymine in each strand. Steady-state and time-resolved analyses revealed that both the FRET efficiency and the fluorescence lifetime of the duplex containing the (6-4) photoproduct were almost identical to those of the undamaged duplex, while marked differences were observed for a cisplatin-modified duplex, as a model of kinked DNA. Lifetime measurements of a series of duplexes containing the (6-4) photoproduct, in which the fluorescein position was changed systematically, revealed a small unwinding at the damage site, but did not suggest a kinked structure. These results indicate that formation of the (6-4) photoproduct induces only a small change in the DNA structure, in contrast to the large kink at the (6-4) photoproduct site reported in an NMR study.
Asunto(s)
ADN/química , Conformación de Ácido Nucleico , Espectrometría de Fluorescencia/métodos , Cisplatino/química , Fluorescencia , Oligonucleótidos/químicaRESUMEN
Communication between nerves and mast cells is a prototypic demonstration of neuroimmune interaction. We used an in vitro co-culture approach comprising cultured murine superior cervical ganglia (SCG) and rat basophilic leukemia (RBL-2H3) cells. Atomic force microscopy (AFM) showed how neurites attached to a pseudopodium or a cell body of an RBL cell. After stimulation of SCG neurites with bradykinin or scorpion venom, RBL cells attached to neurites spread and flattened, and several discharged granules (0. 5-1.0 microm in diameter) were found on the surface of the RBL cells. A neurokinin (NK)-1 receptor (i.e. substance P receptor) antagonist prevented the RBL degranulation. The results showed that activation of the SCG neurites with bradykinin or scorpion venom was able to elicit degranulation in RBL cells which were attached to neurites.