RESUMO
This paper investigates, through simulation and experiment, the behavior of two dimensional foci arrays generated via phase-only holography where an iterative algorithm was used to produce the kinoforms. Specifically, we studied how aliasing of the signal on a spatial light modulator affects the quality of the foci array as the density and size of the array are varied. This study provides a reference for applications where it is important to understand how the fidelity and overall quality of the foci array changes as the number of foci increases and as the spacing between foci decreases.
Assuntos
Holografia/métodos , Algoritmos , Holografia/estatística & dados numéricos , Fenômenos ÓpticosRESUMO
Confocal fluorescence microscopy coupled with a diffraction-limited laser beam and a high-efficiency detection system has been used to study the diffusive movement and emission process of individual fluorescent molecules in the liquid phase at room temperature. The high detection sensitivity achieved at fast data acquisition speeds (greater than 1 kilohertz) allows real-time observation of single-molecule fluorescence without statistical analysis. The results show fluorescence-cycle saturation at the single-molecule level and multiple recrossings of a single molecule into and out of the probe volume as well as the triplet state.
Assuntos
Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Rodaminas/análise , Fluoresceína , Fluoresceínas/análise , Fluorescência , Corantes Fluorescentes/análise , Microscopia Confocal/instrumentação , Microscopia de Fluorescência/instrumentação , Soluções , TemperaturaRESUMO
Secretory vesicles obtained from the atrial gland of the gastropod mollusk Aplysia californica were chemically analyzed individually with a combination of optical trapping, capillary electrophoresis separation, and a laser-induced fluorescence detection. With the use of optical trapping, a single vesicle that had attoliters (10(-18) liters) of volume was introduced into the tapered inlet of a separation capillary. Once the vesicle was injected, it was lysed, and its components were fluorescently labeled with naphthalene-2, 3-dicarboxaldehyde before separation. The resultant electropherograms indicated distinct variations in the contents of single vesicles.
Assuntos
Aminas/análise , Aminoácidos/análise , Grânulos Citoplasmáticos/química , Eletroforese Capilar , Taurina/análise , Animais , Aplysia/química , Aplysia/ultraestrutura , Espectrometria de Massas , Naftalenos , Peptídeos/análise , Cianeto de Potássio , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Individual phospholipid vesicles, 1 to 5 micrometers in diameter, containing a single reagent or a complete reaction system, were immobilized with an infrared laser optical trap or by adhesion to modified borosilicate glass surfaces. Chemical transformations were initiated either by electroporation or by electrofusion, in each case through application of a short (10-microsecond), intense (20 to 50 kilovolts per centimeter) electric pulse delivered across ultramicroelectrodes. Product formation was monitored by far-field laser fluorescence microscopy. The ultrasmall characteristic of this reaction volume led to rapid diffusional mixing that permits the study of fast chemical kinetics. This technique is also well suited for the study of reaction dynamics of biological molecules within lipid-enclosed nanoenvironments that mimic cell membranes.
Assuntos
Bioquímica/métodos , Lipossomos , Fosfatase Alcalina/metabolismo , Cálcio/metabolismo , DNA/metabolismo , Difusão , Eletroquímica , Eletroporação , Fluoresceínas/metabolismo , Fluorescência , Corantes Fluorescentes/metabolismo , Concentração de Íons de Hidrogênio , Bicamadas Lipídicas , Microeletrodos , Microscopia Confocal , Microscopia de Fluorescência , Miniaturização , Técnicas de Patch-Clamp , FosfolipídeosRESUMO
The sickle erythrocyte (RBC) is a pathologic RBC that contains multiple membrane abnormalities. Some of these abnormalities have been implicated in the pathophysiology of vasoocclusive crises characteristic of sickle cell disease; others have yet to be defined in terms of their clinical significance. Recent information has shown that sickle RBC adhere abnormally to cultured endothelial cells yet little is known about the ways in which sickle cells interact with model membranes of defined size and lipid composition. We investigated this phenomenon by interacting sickle RBC with artificial lipid vesicles (liposomes) containing acidic phospholipids. Our results demonstrate that sickle disease (hemoglobin SS) RBC bind more of these liposomes than do normal or sickle trait (hemoglobin AS) RBC and that these differences are accentuated by hypoxia-induced sickling. Binding of liposome phospholipid to sickled RBC was not attributable to phospholipid exchange between liposomes and RBC and was consistent with a mechanism involving both membrane fusion and a stable reversible adhesion of liposomes to the RBC membrane.Investigations into the mechanism(s) underlying increased liposome binding to sickled RBC suggested that the known reversible translocation of aminophospholipids, phosphatidylserine (PS) and phosphatidyl-ethanolamine (PE), from the inner to the outer leaflet of the reversibly sickled RBC (RSC) plasma membrane during sickling may be a component of increased liposome binding to RSC. This idea was supported from results of experiments in which normal RBC were treated with diamide resulting in the expression of outer leaflet PE and PS and a stimulation of liposome binding to these cells. However, sickle RBC separated according to cell density on stractan gradients showed that irreversibly sickled RBC (ISC) were less capable of liposome binding than were discoid RSC. Since ISC are known to contain elevated levels of outer leaflet aminophospholipids, such a result suggests that other changes in the plasma membrane of sickle cells, in addition to phospholipid reorganization, are probably involved in enhanced liposome binding to these cells. In other experiments, we showed that liposomes containing l-phenylalanine were capable of delivering this antisickling agent into intact sickle RBC as demonstrated by the partial inhibition of hypoxia-induced sickling in vitro. Our results suggest that liposomes can be used as sensitive probes for investigating changes in RBC membrane properties, especially those that affect intermembrane interactions, and that liposomal transport systems may have significant implications in the therapy of sickle cell disease.
Assuntos
Anemia Falciforme/sangue , Eritrócitos/metabolismo , Lipossomos/metabolismo , Fosfatidilcolinas/fisiologia , Fosfatidilserinas/fisiologia , Separação Celular , Diamida/farmacologia , Ácido Edético/farmacologia , Eritrócitos/efeitos dos fármacos , Humanos , Oxigênio/farmacologia , Fenilalanina/farmacologia , Surfactantes Pulmonares/metabolismo , Traço Falciforme/sangue , Trioleína/metabolismoRESUMO
Our plan was to evaluate the potentially important role of phospholipids in erythrocyte shape alterations by determining if their orientation was altered during endocytosis. Stomatocytosis and endocytosis were induced in normal intact human erythrocytes by incubation with three agents: primaquine, vinblastine, and chlorpromazine, each of which has its own requirements and time course for producing endocytosis. The organization of the phospholipid bilayer was assessed by measuring the extent of degradation of phophatidylcholine (PC), phophatidylethanolamine (PE), phosphatidylserine (PS), and sphingomyelin (SM) produced by exposure of erythrocytes to a nonpenetrating protease-free phospholipase A2 alone or in combination with a purified sphingomyelinase as well. The induction of stomatocytosis did not change this orientation. However, correlating with the onset of endocytosis but not its extent, there was an increase in PE degradation, which could be detected regularly only by use of phospholipase A2 alone. Use of the combination of phospholipase A2 and sphingomyelinase showed that the extent and course of endocytosis was paralleled by an apparent movement of PC and SM from the outer to the inner half of the lipid bilayer. Since no further PE was hydrolyzed and because no PS was ever degraded, this inward movement of PC and SM did not represent the establishment of complete symmetry in the membrane. By adjusting the experimental design it was possible to implicate the endocytic process, and not insertion of drug in the membrane, as the cause of the alterations in phospholipid organization seen. Our findings indicate that the phospholipid orientation is very closely involved in the endocytosis process and that specific states of phospholipid asymmetry may be related to identifiable membrane events.
Assuntos
Endocitose , Membrana Eritrocítica/metabolismo , Bicamadas Lipídicas/sangue , Clorpromazina/farmacologia , Membrana Eritrocítica/efeitos dos fármacos , Eritrócitos/citologia , Humanos , Fosfatidilcolinas/sangue , Fosfatidiletanolaminas/sangue , Fosfolipases A/farmacologia , Fosfolipases A2 , Fosfolipídeos/sangue , Primaquina/farmacologia , Esfingomielina Fosfodiesterase/farmacologia , Vimblastina/farmacologiaRESUMO
The precise mechanism by which sickle erythrocytes (RBC) are removed from the circulation is controversial, although it is possible that enhanced recognition of these cells by circulating mononuclear phagocytes could contribute to this process. We investigated this possibility by interacting sickle cells with cultured human peripheral blood monocytes. Our results show that both irreversibly sickled cells (ISC) and deoxygenated reversibly sickled cells (RSC) had a higher avidity for adherence to monocytes than did oxygenated sickle and normal RBC. ISC were the most adherent cell type. Adherence of RSC to monocytes was found to be reversible; reoxygenation of deoxygenated RSC resulted in a significant decrease in RSC--monocyte adherence. Concomitant with alterations in sickle RBC adherence were alterations in the organization and bilayer distribution of membrane phospholipids in these cells. Specifically, enhanced adherence was associated with increased exposure of RBC membrane outer leaflet phosphatidylserine (PS) and phosphatidylethanolamine, whereas lack of adherence was associated with normal patterns of membrane phospholipid distribution. To investigate the possibility of whether the exposure of PS in the outer membrane leaflet of these cells might be responsible for their recognition by monocytes, the membranes of normal RBC were enriched with the fluorescent PS analogue 1-acyl-2[(N-4-nitro-benzo-2-oxa-1,3-diazole)aminocaproyl]-phosphatidy lse rine (NBD-PS) via transfer of the exogenous lipid from a population of donor phospholipid vesicles (liposomes). RBC enriched with NBD-PS exhibited enhanced adherence to monocytes, whereas adherence of RBC enriched with similar amounts of NBD-phosphatidylcholine (NBD-PC) was not increased. Furthermore, preincubation of monocytes with PS liposomes resulted in a approximately 60% inhibition of ISC adherence to monocytes, whereas no inhibition occurred when monocytes were preincubated with PC liposomes. These findings strongly suggest that erythrocyte surface PS may be a ligand recognized by receptors on human peripheral blood monocytes and that abnormal exposure of PS in the outer leaflet of the RBC membrane, as found in sickle RBC, might serve to trigger their recognition by circulating monocytes. Our results further suggest that abnormalities in the organization of erythrocyte membrane phospholipids may have significant pathophysiologic implications, possibly including shortened cell survival.
Assuntos
Anemia Falciforme/fisiopatologia , Envelhecimento Eritrocítico , Membrana Eritrocítica/fisiologia , Eritrócitos Anormais/fisiologia , Monócitos/fisiologia , Fosfatidilserinas/sangue , Adesão Celular , Células Cultivadas , Humanos , Lipídeos de Membrana/fisiologia , Microscopia Eletrônica de Varredura , Oxigênio/sangue , Fosfatidilcolinas/sangue , Formação de RosetaRESUMO
We have previously reported that the normal membrane phospholipid organization is altered in sickled erythrocytes. More recently, we presented evidence of enhanced transbilayer movement of phosphatidylcholine (PC) in deoxygenated reversibly sickled cells (RSC) and put forward the hypothesis that these abnormalities in phospholipid organization are confined to the characteristic protrusions of these cells. To test this hypothesis, we studied the free spicules released from RSC by repeated sickling and unsickling as well as the remnant despiculated cells. The rate of transbilayer movement of PC in the membrane of deoxygenated remnant despiculated cells was determined by following the fate of 14C-labelled PC, previously introduced into the outer monolayer under fully oxygenated conditions using a PC-specific phospholipid exchange protein from beef liver. The rate of transbilayer movement of PC in the remnant despiculated cells was significantly slower than in deoxygenated native RSC and was not very much different from that in oxygenated native RSC or irreversibly sickled cells. The free spicules had the same lipid composition as the native cells, but were deficient in spectrin. These spicules markedly enhanced the rate of thrombin formation in the presence of purified prothrombinase (Factor Xa, Factor Va, and Ca2+) and prothrombin, indicating the exposure of a significant fraction of phosphatidylserine (PS) in the outer monolayer. This effect was not observed when the spicules in this assay were replaced by normal erythrocytes, deoxygenated native RSC, or a deoxygenated sample of RSC after repetitive sickling/unsickling. The results are interpreted to indicate that the destabilization of the lipid bilayer in sickled cells, expressed by the enhanced flip-flop of PC and the exposure of PS in the outer monolayer, occurs predominantly in those parts of the membrane that are in spicular form.
Assuntos
Anemia Falciforme/sangue , Membrana Eritrocítica/ultraestrutura , Bicamadas Lipídicas , Fosfolipídeos/fisiologia , Centrifugação , Humanos , Lipídeos de Membrana/análise , Proteínas de Membrana/análise , Fosfatidilcolinas/análise , Fosfatidilserinas/análise , Fosfolipases A/farmacologia , Tromboplastina/metabolismoRESUMO
One constraint of semiconducting polymer dots (Pdots), especially those with near-IR emission, is their low effective emitter ratio (â¼1.5 mole percent), which limits their pH sensing performance. The other critical issue of existing Pdot-based pH sensors is their poor photostability. To address these issues, we developed a series of Pdots by dendronizing the squaraine-based pH responsive near-IR emitter, which is covalently incorporated into the polyfluorene (PFO) backbone. The fluorescence self-quenching of the NIR squaraine emitter was effectively suppressed at a high emitter concentration of 5 mole percent. Through controlling the individually incomplete energy transfer from the amorphous PFO donor to the blue ß-phase PFO and NIR squaraine emitter, we obtained a ratiometric pH sensor with simultaneously improved pH sensitivity, brightness, and photostability. The Pdots showed a fast and reversible pH response over the whole biological pH range of 4.7 to 8.5. Intracellular pH mapping was successfully demonstrated using this ultra-bright and photostable Pdot-based pH indicator.
RESUMO
Nerve injury elicits both universal and limited responses. Among the former is regenerative growth, which occurs in most peripheral neurons, and among the latter is the long-term hyperexcitability that appears selectively in nociceptive sensory neurons. Since positive injury signals communicate information from the site of an injury to the cell body, we hypothesize that a nerve injury activates both universal and limited positive injury signals. Studies in Aplysia indicate that protein kinase G is a limited signal that is responsible for the induction of long-term hyperexcitability. Given that long-term hyperexcitability contributes to chronic pain after axotomy in rodent neuropathic pain models, we investigated its underlying basis in the rat peripheral nervous system. Using biochemical assays, Western blots, and immunocytochemistry we found that the Type 1alpha protein kinase G is the predominant isoform in the rat periphery. It is present primarily in axons and cell bodies of nociceptive neurons, including populations that are isolectin B4-positive, isolectin B4-negative, and those that express transient receptor potential vanilloid receptor-1. Surprisingly, protein kinase G is not present in the facial nerve, which overwhelmingly contains axons of motor neurons. Crushing the sciatic nerve or a cutaneous sensory nerve activates protein kinase G in axons and results in its retrograde transport to the neuronal somata in the DRG. Preventing the activation of protein kinase G by injecting Rp-8-pCPT-cGMPS into the crush site abolished the transport. The protein kinase A inhibitor Rp-8-pCPT-cAMPS had no effect. Extracellular signal-related kinases 42/44 are also activated and transported after nerve crush, but in both motor and sensory axons. Chronic pain has been linked to long-term hyperexcitability following a nerve inflammation in several rodent models. We therefore injected complete Freund's adjuvant into the hindpaw to induce an inflammation and found that protein kinase G was activated in the sural nerve and transported to the DRG. In contrast, the extracellular signal-related kinases in the sensory axons were not activated by the complete Freund's adjuvant. These studies support the idea that the extracellular signal-related kinases are universal positive axonal signals and that protein kinase G is a limited positive axonal signal. They also establish the association between protein kinase G, the induction of long-term hyperexcitability, and chronic pain in rodents.
Assuntos
Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Inflamação/patologia , Neurônios Aferentes/enzimologia , Nociceptores/enzimologia , Neuropatia Ciática/patologia , Animais , Axônios/efeitos dos fármacos , Axônios/enzimologia , Western Blotting/métodos , Contagem de Células , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Inibidores Enzimáticos/farmacologia , Adjuvante de Freund/toxicidade , Gânglios Espinais/patologia , Imuno-Histoquímica/métodos , Inflamação/induzido quimicamente , Masculino , Proteínas de Neurofilamentos/metabolismo , Neurônios Aferentes/patologia , Nociceptores/fisiopatologia , Transporte Proteico/fisiologia , Ratos , Ratos Sprague-Dawley , Proteínas S100/metabolismo , Canais de Cátion TRPV/metabolismo , Tionucleotídeos/farmacologia , Fatores de TempoRESUMO
Historically, it has been theorized that the enhanced oxidant sensitivity of glucose-6-phosphate dehydrogenase (G6PD)-deficient erythrocytes arises as a direct consequence of an inability to maintain cellular glutathione (GSH) levels. This study alternatively hypothesizes that decreased NADPH concentration leads to impaired catalase activity which, in turn, underlies the observed oxidant susceptibility. To investigate this hypothesis, normal and G6PD-deficient erythrocytes and hemolysates were challenged with a H2O2-generating agent. The results of this study demonstrated that catalase activity was severely impaired upon H2O2 challenge in the G6PD-deficient cell while only a transient decrease was observed in normal cells. Supplementation of either normal or G6PD-deficient hemolysates with purified NADPH was found to significantly (P < 0.001) inhibit catalase inactivation upon oxidant challenge while addition of NADP+ had no effect. Analysis of these results demonstrated direct correlation between NADPH concentration and catalase activity (r = 0.881) and an inverse correlation between catalase activity and erythrocyte oxidant sensitivity (r = 0.906). In contrast, no correlation was found to exist between glutathione concentration (r = 0.170) and oxidant sensitivity. Analysis of NADPH/NADPt ratio in acatalasemic mouse erythrocytes demonstrated that NADPH maintenance alone was not sufficient to explain oxidant resistance, and that catalase activity was required. This study supports the hypothesis that impaired catalase activity underlies the enhanced oxidant sensitivity of G6PD-deficient erythrocytes and elucidates the importance of NADPH in the maintenance of normal catalase activity.
Assuntos
Catalase/metabolismo , Eritrócitos/enzimologia , Glucosefosfato Desidrogenase/metabolismo , Peróxido de Hidrogênio/farmacologia , Animais , Membrana Eritrocítica/enzimologia , Eritrócitos/efeitos dos fármacos , Humanos , Camundongos , NAD/metabolismo , NADP/metabolismoRESUMO
To study the effect of sickling on dimyristoylphosphatidylcholine (DMPC)-induced vesiculation, sickle (SS) red blood cells were incubated with sonicated suspensions of DMPC under either room air or nitrogen. Like normal red cells, when sickle cells were incubated with DMPC under oxygenated conditions, incorporation of DMPC into the erythrocyte membrane occurred, followed by echinocytic shape transformation and subsequent release of membrane vesicles. On the other hand, when SS cells were induced to sickle by deoxygenation, DMPC-induced vesiculation of these cells was dramatically reduced. However, upon reoxygenation, release of vesicles from these sickle erythrocytes occurred immediately. When SS cells were incubated under hypertonic (500 mosM) and deoxygenated conditions (where hemoglobin polymerization occurs but red cells do not show the typical sickle morphology), a similar decrease in the extent of vesiculation was observed. Experiments with radiolabelled lipid vesicles indicated that incorporation of DMPC into erythrocyte membranes occurred in all cases and therefore was not the limiting factor in the reduction of vesiculation in deoxygenated SS cells. Taken together, these results indicate that cellular viscosity and membrane rigidity, both of which are influenced by hemoglobin polymerization, are two important factors in process of vesicle release from sickle erythrocytes.
Assuntos
Anemia Falciforme/sangue , Dimiristoilfosfatidilcolina/farmacologia , Eritrócitos/fisiologia , Radioisótopos de Carbono , Dimiristoilfosfatidilcolina/sangue , Membrana Eritrocítica/metabolismo , Eritrócitos/efeitos dos fármacos , Eritrócitos/ultraestrutura , Humanos , Cinética , Microscopia Eletrônica de Varredura , Valores de Referência , Trioleína/sangue , TrítioRESUMO
To provide further understanding of how oxidative damage affects red cell membrane function, the effects of low levels of two different types of oxidants on selected red cell properties have been studied. Hydrogen peroxide (H2O2), an example of a water soluble oxidant, and t-butylhydroperoxide (tBHP), a hydrophobic hydroperoxide, were compared with respect to their effects on membrane permeability, membrane mechanical properties and binding of autologous serum antibodies to the cell surface. Whereas H2O2 treatment resulted in a dose-dependent increase in membrane permeability to potassium that was evident after one hour of oxidant exposure, cells treated with tBHP at doses up to 5 mumol/ml cells showed no immediate change in cation permeability. H2O2 also caused a marked decrease in membrane deformability, whereas tBHP-treated cells showed minimal loss of deformability. However, tBHP treatment did result in a dose-dependent increase in the susceptibility of the membrane to fragmentation under high shear stress. With exclusion of treated samples that bound excess rabbit anti-spectrin antibody, indicating exposure of intracellular components, neither agent promoted the binding of autologous serum antibody in amounts comparable to that found in vivo on high density or some pathologic red cells. Taken together, the results suggest that tBHP and H2O2 cause damage to human red cells by distinct oxidative mechanisms which do not lead directly to substantive generation of binding sites for autologous serum antibodies.
Assuntos
Permeabilidade da Membrana Celular/efeitos dos fármacos , Deformação Eritrocítica/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Peróxidos/farmacologia , Dióxido de Carbono/farmacologia , Membrana Eritrocítica/efeitos dos fármacos , Membrana Eritrocítica/fisiologia , Eritrócitos/imunologia , Eritrócitos/fisiologia , Hemoglobinas/análise , Humanos , Cinética , Fluidez de Membrana/efeitos dos fármacos , Oxirredução , Potássio/sangue , Espectrina/análise , terc-Butil HidroperóxidoRESUMO
Gluthathione peroxidase (gluthatione:hydrogen-peroxide oxidoreductase, EC 1.11.1.9) has been purified approximately 2700-fold from rat lung soluble fraction. The purified enzyme was shown to be homogeneous by sodium dodecyl sulfate/urea polyacrylamide gel electrphoresis. Selenium-75 tracer cochromatographed with the enzyme activity, indicating that rat lung soluble gluthathione peroxidase is a selenium enzyme. The enzyme had an approximate molecular weight of 80000 and contained four identical subunits. The optimal activity of the enzyme was at between pH 8.8 and 9.1. The enzyme had general specificity toward hydroperoxides, and high specificity for reduced glutathione. The kinetic behavior or the purified lung soluble glutathione peroxidase followed a ping-pong-like mechanism; the enzyme first reduced the lipid hydroperoxide substrate to the corresponding hydroxy fatty acid, then was regenerated to the native form by reduced glutathione.
Assuntos
Glutationa Peroxidase/isolamento & purificação , Pulmão/enzimologia , Peroxidases/isolamento & purificação , Animais , Glutationa Peroxidase/metabolismo , Cinética , Masculino , Peso Molecular , Peróxidos , Ratos , Espectrofotometria Ultravioleta , Compostos de SulfidrilaRESUMO
In order to study factors which are involved in maintenance of phosphatidylserine (PS) asymmetry within the human red cell membrane, we measured the effect of ATP-depletion and of membrane skeleton/lipid bilayer uncoupling induced by sickling on the distribution of PS within the membrane bilayer of sickle cells. Trace amounts of radiolabeled PS were introduced into the outer membrane leaflet of both fresh and ATP-depleted reversibly sickled cells (RSCs), using a non-specific lipid transfer protein purified from bovine liver. The equilibration of the newly introduced PS over the two halves of the bilayer was monitored by treatment of the cells with phospholipase A2 which selectively hydrolyzes only those molecules present in the outer membrane leaflet. Within 1 h after insertion into fresh RSCs, only 10% of the labeled PS was accessible to the action of phospholipase A2. This fraction was markedly increased when the cells were subsequently deoxygenated. Prolonged deoxygenation of RSCs, deprived of their ATP after incorporation of radiolabeled PS, caused enhanced phospholipase A2-induced hydrolysis of radiolabeled PS. Similarly, phospholipase A2-induced hydrolysis of endogenous PS in intact RSCs was markedly enhanced when ATP-depleted, but not when fresh cells, were incubated under nitrogen for 3.5 h. Deoxygenated ATP-depleted RSCs markedly enhanced the rate of thrombin formation in the presence of purified coagulation factors Xa, Va, prothrombin and Ca2+. This enhancement appeared to be dependent on the duration of incubation under nitrogen. This phenomenon, indicating the presence of increasing amounts of endogenous PS in the outer membrane leaflet, was not observed when either fresh RSCs or ATP-depleted normal erythrocytes were incubated under nitrogen. Our present observations provide evidence that, in addition to the interaction of PS with the skeletal proteins, an ATP-dependent translocation of PS is required to maintain its absolute asymmetric distribution in the human erythrocyte membrane.
Assuntos
Trifosfato de Adenosina/sangue , Anemia Falciforme/sangue , Membrana Eritrocítica/metabolismo , Bicamadas Lipídicas/sangue , Proteínas de Membrana/sangue , Fosfatidilserinas/sangue , Humanos , Hidrólise , Fosfolipases A/metabolismo , Fosfolipases A2 , Fosfolipídeos/sangue , Tromboplastina/sangueRESUMO
The electrophilic agent, 1-chloro-2,4-dinitrobenzene (CDNB), has been widely used as an intracellular glutathione-depleting agent. However, its possible effect on the functional integrity of cell membrane has largely been neglected. Incubation of human erythrocytes (RBC) with various concentrations of CDNB (0.5 to 5.0 mM) in potassium-free, phosphate buffered saline containing ouabain resulted in a drastic depletion of cellular glutathione as well as a dose-dependent increase in passive potassium leakage. Further, an osmotic gradient ektacytometry profile indicated that the deformability index (DI) of CDNB-treated RBC was substantially lower than the DI value of the control. Also, CDNB caused a dose-dependent increase in the rate of shear-induced fragmentation of resealed ghost prepared from treated, intact erythrocytes. These CDNB-induced changes were accompanied by stomatocytic transformations as evidenced by scanning electron micrographs. Additional study indicated that CDNB caused a dose-dependent decrease in thiol concentrations of RBC membrane. SDS-PAGE analysis of membrane proteins revealed new Coomassie blue stainable bands, most noticeable below band-7 (M.W. 20,000). The effects of CDNB on RBC deformability and membrane proteins were also investigated under an atmosphere without oxygen (under nitrogen) and similar effects were observed between that under room air and that under nitrogen. Taken together, these data strongly indicate that CDNB has an adverse effect on the RBC membrane integrity in addition to its ability to deplete intracellular glutathione, possibly through its interaction with membrane sulfhydryl groups.
Assuntos
Dinitroclorobenzeno/farmacologia , Membrana Eritrocítica/efeitos dos fármacos , Eletroforese em Gel de Poliacrilamida , Deformação Eritrocítica/efeitos dos fármacos , Glutationa/sangue , Humanos , Peróxido de Hidrogênio/farmacologia , Cinética , Proteínas de Membrana/sangue , Oxigênio/farmacologia , Potássio/sangue , Compostos de Sulfidrila/sangueRESUMO
Previous reviews of childhood obesity prevention have focused largely on schools and findings have been inconsistent. Funded by the US Agency for Healthcare Research and Quality (AHRQ) and the National Institutes of Health, we systematically evaluated the effectiveness of childhood obesity prevention programmes conducted in high-income countries and implemented in various settings. We searched MEDLINE®, Embase, PsycINFO, CINAHL®, ClinicalTrials.gov and the Cochrane Library from inception through 22 April 2013 for relevant studies, including randomized controlled trials, quasi-experimental studies and natural experiments, targeting diet, physical activity or both, and conducted in children aged 2-18 in high-income countries. Two reviewers independently abstracted the data. The strength of evidence (SOE) supporting interventions was graded for each study setting (e.g. home, school). Meta-analyses were performed on studies judged sufficiently similar and appropriate to pool using random effect models. This paper reported our findings on various adiposity-related outcomes. We identified 147 articles (139 intervention studies) of which 115 studies were primarily school based, although other settings could have been involved. Most were conducted in the United States and within the past decade. SOE was high for physical activity-only interventions delivered in schools with home involvement or combined diet-physical activity interventions delivered in schools with both home and community components. SOE was moderate for school-based interventions targeting either diet or physical activity, combined interventions delivered in schools with home or community components or combined interventions delivered in the community with a school component. SOE was low for combined interventions in childcare or home settings. Evidence was insufficient for other interventions. In conclusion, at least moderately strong evidence supports the effectiveness of school-based interventions for preventing childhood obesity. More research is needed to evaluate programmes in other settings or of other design types, especially environmental, policy and consumer health informatics-oriented interventions.
Assuntos
Prática Clínica Baseada em Evidências , Obesidade Infantil/prevenção & controle , Saúde Pública , Programas de Redução de Peso , Terapia Comportamental , Criança , Dieta Redutora , Exercício Físico , Comportamento Alimentar , Humanos , Motivação , Obesidade Infantil/epidemiologia , Desenvolvimento de Programas , Estados Unidos/epidemiologia , Programas de Redução de Peso/métodosRESUMO
Oxidative stress has been implicated in protein phosphorylation and dephosphorylation in cells. In our current studies, H2O2 was shown to reversibly inhibit protein tyrosine phosphatase (PTPase) activity in HER14 cells. H2O2 (150 mM) resulted in 40% inhibition of PTPase activity by 15 min and recovery from inhibition was nearly complete by 60 min. H2O2-induced inhibition or recovery of PTPase activity was not affected by cycloheximide, a protein synthesis inhibitor. L-Buthionine-[S,R]-sulfoximine (BSO), an inhibitor of glutathione synthesis, had no effect on H2O2-induced inhibition of PTPase activity but retarded the recovery of activity. Epidermal growth factor (EGF) and EGTA, a Ca2+ chelator, did not influence H2O2-induced inhibition or recovery of PTPase activity. These results suggest that at least 40% of fibroblast PTPase activity can be regulated by cellular redox activity.
Assuntos
Peróxido de Hidrogênio/farmacologia , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Células 3T3/efeitos dos fármacos , Células 3T3/metabolismo , Animais , Linhagem Celular , Cicloeximida/farmacologia , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/metabolismo , Radicais Livres , Humanos , Camundongos , OxirreduçãoRESUMO
Humic acid (HA) has been proposed as a factor that causes Blackfoot disease, an endemic peripheral vascular disease prevailing in the southwest coast of Taiwan. However, the relationship between HA and anemia associated with Blackfoot disease remains unclear. In this study, we showed that HA imposed damages on human red blood cells (RBCs), which were manifested as reduction in deformability of RBCs and hemolysis. At concentrations ranging from 10 to 100 microg/ml, HA caused lipid peroxidation in a dose-dependent manner. Such changes were accompanied by a depletion of glutathione and a reduction in activities of the antioxidant enzymes including catalase, superoxide dismutase, and glucose-6-phosphate dehydrogenase. These results indicate that HA initiates oxidative stress on RBCs and results in their dysfunction. Consistent with our previous findings, the present study supports the notion that HA plays an important role in the pathogenesis of Blackfoot disease.
Assuntos
Anemia/induzido quimicamente , Eritrócitos/efeitos dos fármacos , Substâncias Húmicas/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Doenças Vasculares Periféricas/induzido quimicamente , Deformação Eritrocítica/efeitos dos fármacos , Glutationa/sangue , Hemólise , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , TaiwanRESUMO
Strand breakage of supercoiled pBR322 DNA by a Fenton system is increased in the presence of palladium or platinum (Pt) ions. Neither Pd nor Pt ions can substitute for iron in the Fenton system. We have obtained several lines of evidence that Pd and Pt ions in the presence of a Fenton system can augment the production of OH., as monitored by a spectrophotometric method quantifying hydroxylated salicylate or by a fluorometric method quantifying catechol production. Furthermore, the promoting effect of both metal ions on OH. production was substantiated by the identification of multiple hydroxylated products of salicylate [2,3-dihydroxybenzoate (A), 2,5-dihydroxybenzoate (B), and catechol (C)] using HPLC. The concentrations of A, B, and C produced in the control were 4.5, 8.0, and 2.0 microM, respectively; whereas, their respective concentrations increased to 23.6, 42.0 and 10.0 microM with the addition of Pd ions. The observed phenomenon was further confirmed by the identification of HO-DMPO spin adducts using ESR spectroscopy. Taken together, our data suggest that the mechanism of Pd or Pt ion-mediated exacerbation of DNA damage by a Fenton system is due to the promotion of OH. production by these metal ions.