RESUMO
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.
RESUMO
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.
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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.
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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.
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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.
Assuntos
Moléculas de Adesão Celular/metabolismo , Dermatite Atópica/metabolismo , Imunoglobulinas/metabolismo , Mastócitos/metabolismo , Células Receptoras Sensoriais/fisiologia , Animais , Adesão Celular , Molécula 1 de Adesão Celular , Moléculas de Adesão Celular/fisiologia , Comunicação Celular/fisiologia , Células Cultivadas , Dermatite Atópica/induzido quimicamente , Pavilhão Auricular , Gânglios Espinais/fisiologia , Haptenos/toxicidade , Imunoglobulinas/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Neuritos/fisiologia , Cloreto de Picrila/toxicidade , Venenos de Escorpião/farmacologiaRESUMO
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.
Assuntos
DNA/química , Conformação de Ácido Nucleico , Espectrometria de Fluorescência/métodos , Cisplatino/química , Fluorescência , Oligonucleotídeos/químicaRESUMO
Two-dimensional (2D) crystals of beef liver catalase were prepared by adsorption to a film of synthetic polypeptide, poly(1-benzyl-L-histidine) (PBLH), spread at the air/water interface. The crystallization experiments were carried out in the pH range of 4.8-6.4 for catalase solutions at low concentration (10 micrograms/ml). The pH-dependence suggested an electrostatic interaction in the binding of catalase to the PBLH film. At lower pH, small crystals were formed at a low binding rate, and at higher pH the binding was rapid and densely-packed 2D arrays with poor crystallinity were formed. To stimulate crystal growth, a thermal treatment was applied. One-shot heating of the interfacial catalase-PBLH film to 35-40 degrees C was remarkably effective to form larger 2D crystals. The structure of catalase 2D crystals has been analyzed by Fourier filtering of the transmission electron micrographs. The crystal form is a new one, containing four catalase molecules in the unit cell with lattice parameters of alpha = 187 A, b = 225 A and gamma = 92.8 degrees.
Assuntos
Catalase/química , Peptídeos , Animais , Catalase/ultraestrutura , Bovinos , Cristalização , Cristalografia , Concentração de Íons de Hidrogênio , Processamento de Imagem Assistida por Computador , Microscopia Eletrônica , TemperaturaRESUMO
We examined the effect of the two protein tyrosine kinase inhibitors, alpha-cyano-3-ethoxy-4-hydroxy-5-phenylthiomethylcinnamide (ST638) and herbimycin A, on the activation processes of rat basophilic leukemia (RBL-2H3) cells by cross-linking of IgE receptors. RBL-2H3 cells sensitized with DNP-specific monoclonal IgE antibody were stimulated with multivalent antigen (DNP conjugate of bovine serum albumin). Analysis of phosphotyrosine-containing proteins in their lysates by SDS-PAGE and immunoblotting revealed that these two inhibitors efficiently inhibited the tyrosine phosphorylation of several proteins (32, 42, 56, 66, 72, 92, 150 kDa) including phospholipase C-gamma 1. The inhibitors also caused parallel inhibitions of the histamine release, the formation of inositol 1,4,5-trisphosphate, and the increase in cytosolic calcium ion concentration at the late sustained phase. A digital imaging fluorescence microscopic analysis of antigen-dependent calcium signals in individual cells showed that these two tyrosine kinase inhibitors inhibited the calcium influx from the external medium more powerfully than the mobilization of calcium ion from internal stores. In contrast, the inhibitors did not affect the increase in the cytosolic calcium ion concentration or the histamine release induced by the calcium ionophore A23187. Taken together, our results suggest that tyrosine phosphorylation following antigen stimulation regulates phosphatidylinositol hydrolysis and the influx of extracellular calcium.
Assuntos
Cinamatos/farmacologia , Liberação de Histamina/efeitos dos fármacos , Proteínas Tirosina Quinases/antagonistas & inibidores , Quinonas/farmacologia , Receptores de IgG/fisiologia , Sulfetos/farmacologia , Animais , Anticorpos Monoclonais/farmacologia , Benzoquinonas , Cálcio/metabolismo , Linhagem Celular , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Fura-2 , Inositol 1,4,5-Trifosfato/metabolismo , Cinética , Lactamas Macrocíclicas , Leucemia Basofílica Aguda , Peso Molecular , Fosfoproteínas/isolamento & purificação , Fosfoproteínas/metabolismo , Fosfotirosina , Ratos , Receptores de IgG/efeitos dos fármacos , Rifabutina/análogos & derivados , Células Tumorais Cultivadas , Tirosina/análogos & derivados , Tirosina/análiseRESUMO
OBJECTIVES: We sought to determine whether 6 months of treatment with the angiotensin-converting enzyme (ACE) inhibitor enalapril can improve conduit artery endothelial function in young subjects with insulin-dependent diabetes mellitus (IDDM). BACKGROUND: Endothelial dysfunction is an early event in atherogenesis and has been demonstrated in young subjects with IDDM. ACE inhibitors have been shown to enhance conduit artery endothelial function in animal experiments and in patients with established coronary atherosclerosis, although their effect in IDDM is not known. METHODS: Ninety-one subjects (mean age 30.9 years, range 18 to 44) with stable IDDM but no clinical evidence of vascular disease were randomized to receive enalapril (20 mg once daily) (46 subjects) or placebo (45 subjects) in a randomized, double-blind, parallel-group study. Brachial artery flow-mediated dilation (FMD), an endothelium-dependent stimulus, and response to glyceryl trinitrate (GTN), which acts directly on vascular smooth muscle, were assessed noninvasively by means of high resolution external vascular ultrasound at baseline and after 12 and 24 weeks of treatment. RESULTS: FMD was inversely correlated with total cholesterol (r=0.22, p=0.041) but not with any diabetic variables. Treatment with enalapril had no significant effect on FMD (p=0.67) or response to the endothelial-independent dilator GTN (p=0.45). CONCLUSIONS: These data suggest that impairment of endothelial-dependent dilation in young subjects with IDDM is not improved by treatment with the ACE inhibitor enalapril. This lack of improvement may reflect the complex nature of vascular disease in IDDM, which can affect both endothelial and smooth muscle function.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Diabetes Mellitus Tipo 1/fisiopatologia , Enalapril/farmacologia , Endotélio Vascular/efeitos dos fármacos , Adolescente , Adulto , Arteriosclerose/etiologia , Arteriosclerose/prevenção & controle , Artéria Braquial/fisiologia , Angiopatias Diabéticas/prevenção & controle , Método Duplo-Cego , Feminino , Humanos , Masculino , Análise de Regressão , VasodilataçãoRESUMO
Adenosine triphosphatase from the thermophilic bacterium PS3(TF1) has been studied by solution X-ray scattering. A structural change in TF1 caused by the binding of ADP was observed by examining the difference between the radii of gyration of the unligated and ligated forms. The radius of gyration of the unligated TF1 was found to be 49.5 +/- 0.3 A, and it decreased by approximately 3% after ligation with ADP. The positions and the amplitudes of a subsidiary maximum and a shoulder in the scattering profile showed subtle change on nucleotide binding. The lower limit of the maximum length of TF1 was determined to be 165 A for the unligated form and 150 A for the ligated form. The shape analysis of TF1 was performed by model calculations for simple triaxial bodies or their complexes. Among the various models tested, the one that gave the best fit with the experimental data consisted of seven ellipsoids of revolution; six identical ellipsoids with semi-axes: a = b = 18.5 A and c = 74 A. arranged hexagonally, and the other with a = b = 28 A and c = 45 A, located below the other six on the 6-fold axis. On the basis of this model it was suggested that there is a structural change on ligation with nucleotides, consisting of a shrinkage of the six long ellipsoids by 6% along their major axes.
Assuntos
Adenosina Trifosfatases , Bactérias/enzimologia , Difosfato de Adenosina/metabolismo , Sítios de Ligação , ATPase de Ca(2+) e Mg(2+) , Modelos Químicos , Conformação Proteica , Difração de Raios XRESUMO
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.
Assuntos
Artéria Braquial/fisiologia , Ativação Plaquetária/fisiologia , Fluxo Pulsátil/fisiologia , Adulto , Idoso , Arteriosclerose/sangue , Arteriosclerose/complicações , Arteriosclerose/fisiopatologia , Pressão Sanguínea , Feminino , Citometria de Fluxo , Humanos , Masculino , Pessoa de Meia-Idade , Contagem de Plaquetas , Pletismografia , Valores de Referência , Fatores de Risco , Trombose/sangue , Trombose/etiologia , Trombose/fisiopatologiaRESUMO
Atomic force microscopy (AFM) was used for studying gene transfection mediated by cationic liposomes which contain a cationic cholesterol derivative with a different spacer arm. Cationic liposomes were made by a mixture of one of eight cationic cholesterol derivatives and 1,2-dioleoyl-sn-glycero-3-phosphatidyl ethanolamine (DOPE). AFM images showed that vesicles made of the liposome/DNA complex had various diameters depending on each cationic cholesterol derivative with a different spacer arm. The results showed that the diameter of the liposome/DNA complex was well related to the transfection activity of plasmid pSV2CAT DNA to a cultured cell line (NIH3T3). From the results it was found that the vesicles with moderate diameters (from 0.4 to 1.4 microm) were moste effective for gene transfection of plasmid pSV2CAT DNA into the target cell. Neither smaller vesicles (< 400 nm) nor larger vesicles (> 1.4 microm) were adequate for gene transfection. As the gene transfection by the cationic liposomes was mostly inhibited by wortmannin, an inhibitor of endocytosis, it is suggested that the vesicles with moderate diameters were useful for gene transfection by endocytosis.
Assuntos
Colesterol/análogos & derivados , Transfecção/métodos , Células 3T3 , Aminas , Animais , Cloranfenicol O-Acetiltransferase/biossíntese , Endocitose , Genes Reporter , Vetores Genéticos , Lipossomos , Camundongos , Microscopia de Força Atômica/métodos , Fosfatidiletanolaminas , Proteínas Recombinantes/biossínteseRESUMO
A highly fluorescent mutant form of the green fluorescent protein (GFP) has been fused to the human nuclear factor kappaB (NF-kappaB) p50 and p105 (p50/IkappaB gamma), a precursor protein of NF-kappaB p50. GFP-p50 and GFP-p105 were expressed in monkey COS-7 cells and human HeLa cells. Translocation of these chimeric proteins was observed by confocal laser scanning microscopy. GFP-p50 (without IkappaB gamma) in the transfected cells resided in the nucleus. On the other hand, GFP-p105 (GFP-p50 with IkappaB gamma) localized only in the cytoplasm before stimulation and translocated to the nucleus with stimulant specificity similar to that of native NF-kappaB/IkappaB. In addition, the translocation of NF-kappaB to the nucleus had a distinct lag time (a quiescent time) in the target cells. The lag time lasted 10-20 min after stimulation with hydrogen peroxide or tumor necrosis factor alpha. It was suggested that this might be due to the existence of a limiting step where NF-kappaB is released from NF-kappaB/IkappaB by the proteasome.
Assuntos
Núcleo Celular/metabolismo , Proteínas Luminescentes/metabolismo , NF-kappa B/metabolismo , Substituição de Aminoácidos , Animais , Western Blotting , Células COS , Núcleo Celular/efeitos dos fármacos , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Peróxido de Hidrogênio/farmacologia , Proteínas I-kappa B , Proteínas Luminescentes/biossíntese , Proteínas Luminescentes/genética , Microscopia Confocal , NF-kappa B/biossíntese , Subunidade p50 de NF-kappa B , Precursores de Proteínas/biossíntese , Precursores de Proteínas/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/metabolismo , Cifozoários , Fatores de Tempo , Fatores de Transcrição/biossíntese , Fatores de Transcrição/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
By confocal laser scanning microscopy (CLSM) we have studied the membrane fusion between cationic liposomes and the endosome membranes involved in gene transfection mediated by cationic liposomes. Antisense oligonucleotides were transferred by cationic liposomes with a cationic cholesterol derivative, cholesteryl-3beta-carboxyamidoethylenedimethylamine (I). Cationic liposomes were made by a mixture of the derivative I and DOPE. The intracellular distribution of fluorescein-conjugated antisense oligonucleotides (phosphorothioate) was studied by CLSM. The images showed that the antisense oligonucleotides were preferentially transferred into the nucleus of target cells (NIH3T3, COS-7 and HeLa cells) by the liposomes with derivative I. However, their transfection was completely blocked by nigericin which was able to dissipate the pH gradient across the endosome membranes, although the liposome/DNA complex was found in the cytoplasm of the target cells. This was quite in contrast with the fluorescence images of the target cells treated with wortmannin, an inhibitor of endocytosis. The results suggest that at least two steps are effective for gene transfection mediated by the cationic liposomes with cationic cholesterol derivatives. One is the endocytosis of the liposome/DNA complex into the target cells and the other is the removal of antisense oligonucleotides (plasmid DNAs) from the complex in the endosomes. The latter step was preferentially preceded by the membrane fusion between the cationic liposomes and the endosome membranes at around pH 5.0.
Assuntos
Cátions , Lipossomos , Fusão de Membrana , Transfecção , Células 3T3 , Animais , Células COS , Núcleo Celular/metabolismo , Cloranfenicol O-Acetiltransferase/genética , Ésteres do Colesterol , Endossomos/ultraestrutura , Corantes Fluorescentes , Células HeLa , Humanos , Ionóforos/farmacologia , Lasers , Camundongos , Microscopia Confocal , Nigericina/farmacologia , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/análise , FosfatidiletanolaminasRESUMO
Cationic liposomes are known to be useful tools for gene transfection. However, the relation between transfection efficiency and physicochemical properties of liposomes has not been well understood. Here, we synthesized eight cationic derivatives of cholesterol which contain a tertiary amino head group with a different spacer arm. Transfection of plasmid pSV2CAT DNA into cells was done by cationic liposomes made of a mixture of dioleoylphosphatidylethanolamine (DOPE) and each cationic cholesterol derivative. At the same time we measured zeta potential of cationic liposomes by laser Doppler spectroscopy. The present results indicated that zeta potentials of cationic liposomes were well related to transfection activity of pSV2CAT DNA. This suggested that zeta potential of cationic liposomes is one of important factors which control gene transfection.
Assuntos
Cátions , Colesterol/análogos & derivados , Lipossomos/química , Transfecção , Células 3T3 , Animais , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Colesterol/química , Potenciais da Membrana , Camundongos , Fosfatidiletanolaminas , PlasmídeosRESUMO
Apoptosis, a naturally occurring programmed cell death or cell 'suicide', has been paid much attention as one of the critical mechanisms for morphogenesis and tissue remodeling. Activation of cysteine aspartases (caspases) is one of the critical steps leading to apoptosis. Although a mitochondria-mediated pathway has been postulated to be one of the activation mechanism of caspase-3, another subcellular compartment might be involved in the activation of the enzyme. The present study shows that the supernatant fraction of digitonin-treated lysosomes strongly activates Ac-DEVD-CHO inhibitable caspase-3-like protease. Activation of caspase-3-like protease by digitonin-treated lysosomal fractions was specifically suppressed by leupeptin and E-64, inhibitors of cysteine protease. These results indicate that leakage of lysosomal cysteine protease(s) into the cytosolic compartment might be involved in the activation of caspase-3-like protease.
Assuntos
Apoptose , Caspases , Cisteína Endopeptidases/metabolismo , Digitonina/farmacologia , Lisossomos/enzimologia , Animais , Caspase 3 , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Precursores Enzimáticos/metabolismo , Indicadores e Reagentes/farmacologia , Lisossomos/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
The mechanism of the lymphocyte stimulatory action of sulfhydryl group-reactive mercuric ions was studied with respect to its potential ability to induce a protein tyrosine phosphorylation-linked signal for mobilization of free Ca2+ into cytoplasm and nucleus of the cell. Exposure of human leukamic T cell line (Jurkat) cells to high (1 mM) and low (0.01 mM) concentrations of HgCl2 induced tyrosine phosphorylation of multiple proteins in a concentration-dependent manner. Confocal microscopy directly visualized the time course localization of Ca2+ inside the cells after exposure to HgCl2. The onset and level of Ca2+ mobilization following HgCl2 exposure were in parallel to those of protein tyrosine phosphorylation. Interestingly, by either concentration of HgCl2, Ca2+ was mobilized in both cytoplasm and nucleus almost simultaneously, and the level of Ca2+ mobilization in the nucleus was more than that in the cytoplasm. All the HgCl2-mediated Ca2+ mobilization was prevented by addition of protein kinase inhibitor staurosporin prior to HgCl2. These results suggest that heavy metal stress triggers a protein tyrosine phosphorylation-linked signal that leads to a nuclear event-dominant Ca2+ mobilization.
Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Cloreto de Mercúrio/toxicidade , Proteínas/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Inibidores Enzimáticos/farmacologia , Radicais Livres/metabolismo , Humanos , Células Jurkat , Cloreto de Mercúrio/administração & dosagem , Microscopia Confocal , Fosforilação , Proteína Quinase C/antagonistas & inibidores , Estaurosporina/farmacologiaRESUMO
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.
Assuntos
Comunicação Celular , Mastócitos/fisiologia , Microscopia de Força Atômica , Neuritos/fisiologia , Pseudópodes/fisiologia , Animais , Teste de Degranulação de Basófilos , Bradicinina/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas/efeitos dos fármacos , Grânulos Citoplasmáticos/metabolismo , Leucemia Basofílica Aguda/patologia , Mastócitos/metabolismo , Mastócitos/ultraestrutura , Camundongos , Camundongos Endogâmicos CBA , Neuritos/efeitos dos fármacos , Neuritos/ultraestrutura , Pseudópodes/ultraestrutura , Ratos , Venenos de Escorpião/farmacologia , Gânglio Cervical Superior/citologia , Gânglio Cervical Superior/efeitos dos fármacos , Células Tumorais Cultivadas/fisiologia , Células Tumorais Cultivadas/ultraestruturaRESUMO
A confocal fluorescence microscope was used to study the exocytotic secretory processes of mast cells in combination with an fluorescent molecular rotor, 9-(dicyanovinyl)julolidine (DCVJ). DCVJ is known to be an unique fluorescent dye which increases its quantum yield with decreasing intramolecular rotation. Here, DCVJ-loaded peritoneal rat mast cells were stimulated with compound 48/80 and their fluorescence images were compared with fluorescence calcium images of fluo-3-loaded mast cells. Subsequent to transient increases in intracellular free calcium ion concentration, DCVJ fluorescence increased dramatically in the cytoplasm and formed a ring-like structure around the nucleus, suggesting the possibility that the dye bound to the proteins composing the cytoskeletal architecture. Furthermore, the increases of DCVJ fluorescence intensities were mostly blocked in the presence of cytochalasin D (10 microM). However, fluo-3 fluorescence intensities still increased after addition of compound 48/80.
Assuntos
Corantes Fluorescentes , Mastócitos/metabolismo , Nitrilas , Quinolizinas , Compostos de Anilina , Animais , Citocalasina D/farmacologia , Técnicas In Vitro , Cinética , Mastócitos/efeitos dos fármacos , Microscopia de Fluorescência , Ratos , Ratos Wistar , Xantenos , p-Metoxi-N-metilfenetilamina/farmacologiaRESUMO
Eighteen patients with heart failure were studied to clarify whether angiotensin-converting enzyme inhibitor treatment improves excess ventilation during exercise. Treatment with angiotensin-converting enzyme inhibitors had a beneficial effect on excess ventilation during exercise, without significant improvement in exercise capacity in patients with moderate congestive heart failure.