RESUMO
The dispersity, size, and self-interaction of generation 5 polyamidoamine dendrimeric polymers with different terminal groups (surfaces) were characterized using several physicochemical techniques. Amino-surface dendrimers form oligomeric aggregates in aqueous solution, even in the presence of high salt concentrations (0.6M sodium phosphate). In contrast, the hydroxyl-surface polymer G5-OH behaves as a single homogeneous (or paucidisperse) species at low concentration. Measurements of density increment and the sedimentation and diffusion coefficients of G5-OH suggest a more swollen, porous structure than a globular protein of comparable mass. Measurements of the concentration dependence of sedimentation equilibrium of G5-OH in pH 7.2 phosphate buffer indicate the presence of significant electrostatic repulsion overlaid on weakly attractive interactions, leading to the formation of nonspecific aggregates at sufficiently high dendrimer concentration.
Assuntos
Poliaminas/química , Materiais Biocompatíveis/química , Centrifugação com Gradiente de Concentração , Cromatografia em Gel , Dendrímeros , Eletroforese em Gel de Poliacrilamida , Conformação Molecular , Peso Molecular , Tamanho da PartículaRESUMO
Sedimentation equilibrium studies of dilute solutions of tryptophan synthase reveal dissociation from the holoenzyme form, alpha 2 beta 2, into mixtures of alpha beta 2, small amounts of beta 2, and alpha as well as the original alpha 2 beta 2 holoenzyme. The holoenzyme form is stabilized by pyridoxal 5'-phosphate. A new sedimentation equilibrium analytical procedure shows the dissociation of the second alpha subunit to be negatively cooperative. The analytical procedure calculates theoretical error profiles with assumed values of the dissociation constant, k, and a cooperativity parameter until a match is made between one of the theoretical profiles and that computed from experimental data. The latter profile is calculated with an experimentally determined k and assumed values of the cooperativity parameter.
Assuntos
Coenzimas/metabolismo , Fosfato de Piridoxal/metabolismo , Triptofano Sintase/metabolismo , Estabilidade Enzimática , Guanidinas/metabolismo , Holoenzimas/metabolismo , Isotiocianatos/metabolismo , Cinética , Salmonella typhimurium/enzimologia , Estatística como Assunto , Termodinâmica , UltracentrifugaçãoRESUMO
A commercial surface plasmon resonance biosensor, BIACORE X, is employed as a detector in a closed loop of a small sample volume. The sample is continuously circulated by an external syringe pump over two sensor spots, one functionalized with immobilized binding sites to a soluble binding partner in the mobile phase and one serving as a reference surface. A binding isotherm for the interacting macromolecules can be obtained by a stepwise titration of the soluble reactant into the circulating loop, each step followed by observation of the signal increase until equilibrium is attained. Binding constants can be measured under conditions free of mass transport artifacts and without the requirement for regeneration of the immobilized binding sites. This procedure is similar to the stepwise titration procedure described for the cuvette-based sensor design (D. R. Hall and D. J. Winzor, 1997, Anal. Biochem. 244, 152-160). In the presented configuration, the high baseline stability of the instrument combined with the availability of a reference surface for the detection of nonspecific binding permits refractive index changes upon addition of the aliquots to be measured, as well as accounting for temperature or instrumental drifts, and allows for a very long experimental time. This feature extends the applicability of equilibrium titration to systems with higher affinity or slower dissociation rate constants. Furthermore a solution competition titration is described that avoids artifacts from the immobilization procedure to provide a method for measurement of binding constants in solution. Kinetic information on the complex dissociation can also be obtained by combination of sample delivery via the external pump with the injection of competitor via the microfluidics of the biosensor. The rapid injection of high concentrations of competitor allows the observation of fast dissociation processes under conditions minimizing rebinding.
Assuntos
Técnicas Biossensoriais , Ligação Proteica , Animais , Anticorpos/imunologia , Cromatografia de Afinidade , Fragmentos Fab das Imunoglobulinas/imunologia , Cinética , Mioglobina/imunologia , Neuraminidase/imunologia , Ressonância de Plasmônio de Superfície , BaleiasRESUMO
A new method is described that allows measurement of the molar mass of the solute within 15 to 30 min after start of a conventional long-column sedimentation equilibrium experiment. A series of scans of the concentration distribution in close vicinity of the meniscus, taken in rapid succession after the start of the centrifuge run, is analyzed by direct fitting using the Lamm equation and the Svedberg equation. In case of a single solute, this analysis of the initial depletion at the meniscus reveals its buoyant molar mass and sedimentation coefficient with an accuracy of approximately 10% and provides gross information about sample heterogeneity. This method can be used to study macromolecules that do not possess the prolonged stability needed in conventional sedimentation equilibrium experiments and it can increase the efficiency of sedimentation equilibrium experiments of previously uncharacterized samples.
Assuntos
Química/métodos , Modelos Teóricos , Proteínas/química , Enzimas/química , Peso Molecular , Soluções , Termodinâmica , Fatores de TempoRESUMO
Pelleting and resuspension of Fura 2-labeled c6 glioma cells leads to a large Ca flux characterized by a high initial internal level of Ca which rapidly declines to close to basal levels. The effect has been termed resuspension-induced ion flux (RIIF). The RIIF effect is temperature dependent and requires external calcium, cytoskeletal integrity, and functional calcium and potassium channels. The magnitude of the RIIF effect is dependent upon pelleting speed, suggesting cell contact and reduction in external fluid medium to be important causative parameters. Several other cell species also exhibit the RIIF effect.
Assuntos
Cálcio/metabolismo , Animais , Canais de Cálcio/metabolismo , Técnicas de Cultura de Células , Linhagem Celular , Centrifugação , Quelantes/farmacologia , Colchicina/farmacologia , Ácido Egtázico/farmacologia , Corantes Fluorescentes , Fura-2 , Humanos , Transporte de Íons/efeitos dos fármacos , Canais de Potássio/metabolismo , Ratos , TemperaturaRESUMO
Beta-endorphin and naloxone bind to Jurkat cell membrane preparations and can mutually displace each other from membrane binding sites. Tetraethylammonium ion, a potassium channel blocker, competitively displaces beta-endorphin and naloxone from membrane binding sites. Mitogen stimulated calcium ion flux is inhibited by tetraethyl ammonium and this inhibition is relieved by naloxone. With data derived from whole cell calcium ion flux studies, we accurately calculated the competitive displacement of beta-endorphin and naloxone from membrane preparations by tetraethylammonium thus showing that the action of these agents on potassium channels does not require second messengers. Using the resuspension induced ion flux technique, we find that beta-endorphin competes against naloxone for binding to Jurkat cells and naloxone competes against charybdotoxin, a potassium channel inhibitor, which like tetraethylammonium, is known to bind to the outer vestibule of the channel. Patch clamp electrophysiological studies show that beta-endorphin and naloxone exert complex actions on potassium channels in the presence or absence of mitogens. We conclude that one molecule of beta-endorphin or naloxone, but not both at the same time, bind to an area near the charybdotoxin/tetraethylammonium binding locus of Jurkat potassium channels.
Assuntos
Naloxona/metabolismo , Antagonistas de Entorpecentes/metabolismo , Bloqueadores dos Canais de Potássio , Linfócitos T/metabolismo , Compostos de Tetraetilamônio/metabolismo , beta-Endorfina/metabolismo , Sítios de Ligação , Ligação Competitiva , Bloqueadores dos Canais de Cálcio/farmacologia , Charibdotoxina/farmacologia , Eletrofisiologia , Mitógenos/farmacologia , Naloxona/farmacologia , Antagonistas de Entorpecentes/farmacologia , Canais de Potássio/efeitos dos fármacos , Canais de Potássio/fisiologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Compostos de Amônio Quaternário/farmacologia , Tetraetilamônio , Compostos de Tetraetilamônio/farmacologia , Células Tumorais Cultivadas , beta-Endorfina/farmacologiaRESUMO
The dissociation equilibria of Salmonella typhimurium tryptophan synthase alpha 2 beta 2 complex were studied via centrifugation of the complex to sedimentation equilibrium in neutral saline buffers containing 0 to 137 mM guanidine isothiocyanate (GuSCN). The resulting concentration gradients were analyzed in the context of an equilibrium model for sequential dissociation of two alpha subunits from a stable beta 2 subunit. Under the conditions of these experiments, the first dissociation constant alone could be evaluated at GuSCN concentrations < or = 100 mM, and the second dissociation constant alone could be evaluated at GuSCN = 137 mM. At intermediate GuSCN, both dissociation constants were sufficiently well defined to rule out the presence of a large equilibrium cooperative effect in the stepwise dissociation of the alpha subunits.
Assuntos
Proteínas de Bactérias/química , Guanidinas/química , Isotiocianatos/química , Triptofano Sintase/química , Proteínas de Bactérias/metabolismo , Soluções Tampão , Centrifugação , Cinética , Substâncias Macromoleculares , Computação Matemática , Salmonella typhimurium/enzimologia , Cloreto de Sódio , Triptofano Sintase/metabolismoRESUMO
10(-6) M n-formyl-methionyl-leucyl-phenylalanine (FMLP) stimulated Ca2+ flux in human neutrophils is characterized by a profile composed of two peaks of different amplitude and breadth. beta-Endorphin inhibited the magnitude and modulated the kinetics of the second peak in a manner which was dose-dependent and could reflect either negative cooperativity or heterogeneity of binding sites. The second peak arises from calcium channel activity since in the presence of nifedipine or EGTA it was not evident while the first peak was reduced about 24%. Similarly, at 15 degrees C, where we were unable to detect any channel activity, the first peak was diminished by 35% and beta-endorphin had no detectable effect on this peak. These results led us to conclude that the first peak is chiefly composed of Ca2+ recruited from cytosolic stores which are relatively insensitive to the above treatments and a smaller fraction of calcium originating in calcium channel activity. Hence, we reason that beta-endorphin modulates only the calcium ion flux arising from calcium channel function.
Assuntos
Canais de Cálcio/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , beta-Endorfina/farmacologia , Cálcio/metabolismo , Canais de Cálcio/fisiologia , Citosol/metabolismo , Ácido Egtázico/farmacologia , Humanos , Neutrófilos/metabolismo , Nifedipino/farmacologia , TemperaturaRESUMO
Natural killer (NK) cell activity and mitogen-stimulated spleenocyte proliferation were measured in rats exposed to stress in the form of avoidable and unavoidable shock. Rats that could avoid shock exhibited higher NK activity than either unshocked controls or rats that could not avoid shock. The latter were yoked to the avoidance rats and thus received the same number and frequency of shocks as did the avoidance group. The increased NK activity in the avoidance group appears due to a higher number of NK cells in this group as compared with those in the control or unavoidable shock groups. Additionally, NK activity was found to be proportional to avoidance response rate, with a majority of animals exceeding the minimal temporal avoidance requirement. Mitogen-stimulated proliferation of spleenocytes was also increased several fold in the group that could avoid shock as compared with that which could not and controls. The difference in NK activity and mitogen-stimulated proliferation could not be ascribed to differences in cortisol levels. The results indicate that behavior which results in the avoidance of aversive stimuli can lead to significant enhancement of immune system competence.
Assuntos
Aprendizagem da Esquiva , Citotoxicidade Imunológica , Células Matadoras Naturais/imunologia , Ativação Linfocitária , Linfócitos T/imunologia , Animais , Eletrochoque , Imunocompetência , Ativação Linfocitária/genética , Masculino , Ratos , Estresse Fisiológico/imunologiaRESUMO
We find that beta-endorphin (Bend) can have, positive, negative, or neutral dose-dependent effects on the mitogen-stimulated proliferation of human peripheral blood lymphocytes. The distribution of positive, negative, or neutral responses was nonrandom. In studies carried out over a year, we show that an individual's mitogen-stimulated lymphocyte proliferative response to Bend can change with time. We show that the inhibition induced by cortisol can be, in part, relieved by Bend. On the basis of our results and those of others in the field, we put forward a model that can qualitatively account for many of the observations we and other investigators have made.
Assuntos
Ativação Linfocitária/efeitos dos fármacos , Neuroimunomodulação , beta-Endorfina/farmacologia , Adulto , Relação Dose-Resposta Imunológica , Feminino , Humanos , Hidrocortisona/antagonistas & inibidores , Hidrocortisona/farmacologia , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Neuroimunomodulação/fisiologia , Fito-Hemaglutininas/farmacologia , Linfócitos T/efeitos dos fármacos , Fatores de TempoRESUMO
To characterize the effect of beta-endorphin on T-lymphocyte activation, we examined its influence on membrane currents, intracellular calcium flux, and c-myc mRNA levels during mitogenic stimulation of Jurkat cells. While beta-endorphin weakly enhanced voltage-activated K+ currents of Jurkat cells by itself, it suppressed these currents in the presence of mitogen. Naloxone, by itself, also enhanced K+ current amplitude, but in the presence of mitogen partially reversed the suppressive effect of beta-endorphin. A 5-30 min exposure to beta-endorphin resulted in an increase in the rate of mitogen-stimulated intracellular calcium release and an increase in c-myc mRNA levels relative to controls. Longer exposure (1-2 h) to beta-endorphin retarded intracellular calcium release, and suppressed c-myc expression. The suppressive effects were reversed by naloxone and mimicked by the K+ channel blocker, tetraethylammonium ion. These data suggest that opiate receptors and K+ channels of Jurkat cells are functionally coupled in a way that modulates intracellular calcium release and c-myc expression - two key processes in T-cell mitogenesis.
Assuntos
Cálcio/metabolismo , Canais de Potássio/efeitos dos fármacos , Proteínas Proto-Oncogênicas/genética , Proto-Oncogenes , Linfócitos T/efeitos dos fármacos , beta-Endorfina/farmacologia , Linhagem Celular , Humanos , Potenciais da Membrana , Naloxona/farmacologia , Proteínas Proto-Oncogênicas c-myc , RNA Mensageiro/análise , Linfócitos T/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Compostos de Tetraetilamônio/farmacologiaRESUMO
Several fluorescent probes have been used in the past to monitor and to measure intracellular calcium and calcium fluxes. The most widely used of these probes are those developed by Tsien. We address the markedly different values obtained when comparing Quin-2 (the original probe) with Fura-2 (a second-generation probe). In most cases the values for intracellular calcium have been considered to be interchangeable for the different probes. Using several different hematopoietic cell lines we show that in no case do the two probes yield equivalent values.
Assuntos
Aminoquinolinas , Benzofuranos , Cálcio/análise , Corantes Fluorescentes , Neutrófilos/metabolismo , Fura-2 , Humanos , Cinética , Células Tumorais CultivadasRESUMO
We compared the effects of prostaglandin D2 (PGD2), prostaglandin F2 alpha (PGF2) and various ketones on superoxide (OX) release by human neutrophils, which had been stimulated by N-formyl methionyl leucyl phenylalanine (FMLP). Our data suggested that the ring carbonyl of PGD2 is essential to its inhibitory effect on OX release, but the carbonyl group as a ketone, alone is not sufficient. Using the fluorescent Ca2+ probe, Fura-2AM, we found that PGD2 increased the rate of decline of FMLP stimulated intracellular free Ca2+ (Ca)i, but that PGF2 had no effect. cAMP altered FMLP stimulated (Ca)i, in a pattern similar to PGD2. Furthermore, the ring carbonyl of PGD2 is crucial to its effect on OX as well as on (Ca)i.
Assuntos
Cálcio/metabolismo , Neutrófilos/efeitos dos fármacos , Prostaglandina D2/farmacologia , Superóxido Dismutase/metabolismo , Humanos , Cetonas/farmacologia , Cinética , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/enzimologia , Neutrófilos/metabolismoRESUMO
Monoiodinated human beta-endorphin was found to bind specifically to human erythrocytes. Unlabeled beta-endorphin and beta-endorphin inhibited binding, but (-)naloxone, [D-Ala2, D-Leu5]-enkephalin, and leu- and met-enkephalin did not. Immunoelectron microscopy, using rabbit anti-beta-endorphin antibody, an antirabbit IgG secondary antibody, and complexed horseradish peroxidase, revealed that at low concentrations beta-endorphin binds to the cell surface. Electron spin resonance spectroscopy showed no effect of beta-endorphin on membrane fluidity. This receptor does not appear to conform to the characteristics of an opiate receptor.
Assuntos
Eritrócitos/metabolismo , Receptores Opioides/metabolismo , beta-Endorfina/metabolismo , Humanos , Fluidez de MembranaRESUMO
Axotomy of a major cranial post ganglionic branch of the superior cervical ganglion of an adult rat resulted in increased radiolabeling of tubulin and microtubule-associated proteins after an in vitro 5-h incubation with [14C]-methionine. The increased incorporation of the radiolabel began at day 1 postoperatively and exhibited a biphasic response, peaking at days 3 and 8.
Assuntos
Gânglios Simpáticos/fisiologia , Regeneração Nervosa , Tubulina (Proteína)/biossíntese , Animais , Eletroforese em Gel de Poliacrilamida , Gânglios Simpáticos/metabolismo , Masculino , Ratos , Ratos EndogâmicosRESUMO
Exposure of microtubular protein to ultraviolet light inhibits its assembly into morphologically normal microtubules. This effect appeared to result primarily from damage to the tubulin dimers. The damage consisted of a conformational change, a loss of two free sulfhydryl groups, a production of higher molecular weight cross-linked species, and the formation of aggregated amorphous material upon polymerization.
Assuntos
Microtúbulos/efeitos da radiação , Raios Ultravioleta , Animais , Encéfalo/ultraestrutura , Bovinos , Dicroísmo Circular , Interfase , Microscopia Eletrônica , Nefelometria e Turbidimetria , Polímeros/metabolismo , Ésteres do Ácido Sulfúrico/metabolismo , Tubulina (Proteína)/efeitos da radiação , UltracentrifugaçãoRESUMO
The effect of 2,450-MHz pulsed microwave radiation on the enzyme activity of membrane-free acetylcholinesterase was studied while the enzyme was in the microwave field. We found no significant effect of microwave radiation on enzyme activity using a wide variety of power densities, pulse widths, repetition rates, and duty cycles. This suggests that simple, direct modification by microwave energy of acetylcholinesterase structure and enzymic activity is not related to microwave alteration of acetylcholinesterase central nervous system levels.