Phosphatidylethanol inhibits phosphatidylinositol-phospholipase C activity in a competitive manner with phosphatidylinositol-4,5-bisphosphate

An obvious difficulty of the study of binding of volatile anesthetics to proteins is to prevent loss of the ligand during the procedure. A novel NMR tube was designed that consists of concentric double cylinders which slide each other under sealed condition. A gas space is left in the tube to measure the free anesthetic concentration in the gas phase, which is in equilibrium with the solution. The enthalpy change of anesthetic transfer from water to BSA, deltaH(w-->r) was -40 kJ x mol(-1). The Gibbs free energy deltaG(w-->r) was -14.0 kJ x mol(-1) at 283 K (K(D) = 2.6 mM) and increased to -11.6 kJ x mol(-1) at 310 K (K(D) = 10.9 mM). The maximum binding site (Bmax) was 19.3 at 10 degrees C and increased to 34.5 at 37 degrees C. The entropy change, deltaS(w-->r) was -92 J x mol(-1) x K(-1) and was almost constant in the temperature range 10 approximately 37 degrees C. Contrary to the general consensus that hydrophobic interaction is entropy-driven, the binding of halothane to BSA was enthalpy-driven, compensating the opposing effect of deltaS with negative deltaH at the biologically meaningful temperature range. Possible cause of the negative deltaS relating to the conformational change of BSA is discussed.     

Protein-facilitated export of arachidonic acid from pig neutrophils

Activated neutrophils release a variety of eicosanoids into the extracellular medium including arachidonic acid, 5-hydroxyicosatetraenoic acid, and leukotriene A4 and B4. In this study, the mechanism of arachidonic acid export has been examined using inside-out plasma membrane vesicles from pig polymorphonuclear leukocytes. Tritiated arachidonic acid associated rapidly with the membrane vesicles and crossed the membrane into the intravesicular space in a time-dependent and saturable manner. Half the maximal influx rate was measured at an arachidonate concentration of 5.7 microM, and a maximal influx velocity of 3.0 nmol/mg x min was determined at pH 6.8. Influx into vesicles was sensitive to a number of common anion transport inhibitors including pentachlorophenol, phloretin, diiodosalicylic acid, and quercetin as well as to the proteases trypsin and Pronase, suggesting a protein-dependent process. Furthermore, influx was temperature-sensitive with an energy of activation of 11.6 kcal/mol. Varying extravesicular concentration of ATP, Na+, or K+ had no impact on arachidonate influx, whereas changes in pH had a profound effect; optimum transport activity was observed at an extravesicular pH of 6, whereas raising the pH to 9.5 essentially abolished uptake. These results indicate and initially characterize a novel protein-facilitated arachidonate export mechanism in pig neutrophils.     

The effects of photodynamic therapy on human neutrophil migration using bacteriochlorin a

Bacteriochlorin a photodynamic therapy (BCA-PDT) caused inhibition of interleukin (IL)-8-activated neutrophil migration, at concentrations that did not induce membrane damage. Random migration and migration induced by other chemoattractants were also inhibited, indicating that the effect of BCA-PDT was not at the level of chemoattractant-receptor interaction but down stream. The BCA-PDT completely blocked superoxide production of phorbol ester-stimulated neutrophils indicating that superoxide production by neutrophils present in the tumor before and during BCA-PDT is not the cause of inactivation of tumor cells. Both type I and type II quenchers prevented inhibition by BCA-PDT but only in electroporated cells. Confocal laser scanning microscopy showed that the fluorescence of BCA was located inside the cell. These results show that the effects of BCA-PDT are intracellular and of a mixed type I/type II character and that the neutrophils present in the tumor during illumination probably do not contribute to tumor eradication by releasing reactive oxygen species.     

Study of poly-L-lysine conformations in aqueous methanol solution by using polarized Raman techniques

Raman polarization measurements of the amide I band are reported in ionized poly-L-lysine dissolved in aqueous methanol. The observed changes with methanol concentration, attributed to changes in coil conformation and to the helix-coil transition, represent a novel method of measuring polymer conformation. Polarization measurements as a function of temperature yield values of the energy differences between rotational isomeric states in the coil. deltaH, of 8.8 +/- 0.7, 10.4 +/- 1.1 and 10.8 +/- 1.5 kJ/mol at methanol concentrations (v/v) of 85, 80 and 70% respectively. The stabilization energy of the helix is estimated at 9.3 kJ/mol.     

Increased saturated triacylglycerol levels in plasma membranes of human neutrophils stimulated by lipopolysaccharide

Neutrophils isolated from patients with bacterial infections or stimulated in vitro with lipopolysaccharide (LPS) produce a high resolution, lipid-dominated spectrum on 1H-NMR spectroscopy (May et al, 1993. J. Infect. Dis. 168: 386-392). We have investigated the origin of this lipid signal using NMR and chemical analyses of both whole neutrophils and purified plasma membranes. Plasma membranes from neutrophils that had been stimulated with 50 microg/ml LPS exhibited the high resolution 1H-NMR signal, and contained double the triacylglycerol (TAG) content of plasma membranes isolated from resting cells. Chemical analysis of the whole cells indicated that the TAG also increased at the cellular level (1.7-fold) after stimulation with LPS. Diradylglycerol increased 2- to 3-fold in both whole cells and plasma membranes after stimulation, but was only a minor component compared with TAG. The plasma membrane protein/phospholipid ratio increased 2.6-fold, whereas cholesterol (free and esterified) was unchanged. The membranes from LPS-stimulated neutrophils exhibited increased fluidity, as judged by increased merocyanine 540 binding, consistent with a 2-fold reduction in cholesterol/phospholipid ratio. LPS induced a shift in fatty acid content of whole cell polar lipids towards more oleic acid and less palmitic acid, whereas the neutral lipid fraction contained increased amounts of palmitic and stearic acids. The TAG fraction of plasma membrane lipids contained increased amounts of palmitic acid when prepared from cells stimulated with LPS. We conclude that the 1H-NMR signal in LPS-stimulated neutrophils arises from increased amounts of plasma membrane TAG with an elevated content of palmitic acid.     

Platelet-activating factor induces a concentration-dependent spectrum of functional responses in bovine neutrophils

We characterized the dose response of bovine neutrophils to platelet-activating factor (PAF) with respect to the following functions: calcium flux and membrane potential changes, actin polymerization, degranulation, and the production and/or priming of the oxidative burst. PAF at very low concentrations (10(-10) and 10(-9) M) caused changes in intracellular calcium and membrane potential in bovine neutrophils, whereas moderate PAF concentrations (> or = 10(-7) M) resulted in increased actin polymerization. Degranulation responses to PAF were more complex: low concentrations (10(-9) M) caused secretory granule degranulation, moderate doses (> or = 10(-7) M) caused specific granule degranulation, whereas azurophil degranulation only occurred at high (10(-5) M) PAF concentrations. Treatment of bovine neutrophils with PAF at concentrations > or = 10(-7) M also caused up-regulation of the adhesion molecules Mac-l and L-selectin. PAF stimulation resulted in a very weak [compared to phorbol myristate acetate (PMA)] oxidative burst in bovine neutrophils, and only at high (10(-6) M) concentrations. Unlike human neutrophils, bovine neutrophils were poorly primed by PAF treatment. Only high concentrations of PAF (10(-5) M) caused an increased rate of PMA-stimulated superoxide production, although lower doses of PAF did reduce the lag time preceding the PMA-induced oxidative burst. The overall pattern that can be inferred is that lower concentrations of PAF promote neutrophil sensitivity and interaction by selective degranulation, up-regulation of adhesion molecules, and increased actin polymerization. In contrast, higher PAF concentrations can promote, albeit weakly, more direct bactericidal responses, such as the release of reactive oxygen species and granule enzymes. The ability of PAF to modulate a graded response in bovine neutrophils would allow the cell to respond proportionally to the severity of a stimulus.     

荧光猝灭法与紫外-可见吸收光谱法相结合探讨纳米MnS与明胶相互作用

利用荧光光谱、同步荧光光谱和紫外-可见吸收光谱研究了在pH 12.0及不同温度下MnS纳米晶与明胶结合反应的光谱行为。结果表明, 在明胶溶液中生成的MnS纳米晶与明胶通过范德华和氢键作用力结合成复合物, 使明胶的内源荧光猝灭(属于静态荧光猝灭), 而MnS纳米晶却未使明胶色氨酸残基的构象发生变化。根据静态猝灭的 Lineweave-Burk 方程, 计算了温度在293 K和305 K时MnS纳米晶与明胶的结合常数和热力学参数, 得到结合常数分别为4.72×10³L·mol^-1和3.23×10³L·mol^-1, 焓变(ΔH)为-23.5 kJ·mol^-1, 熵变(ΔS)分别为-9.86 J·K^-1·mol^-1和-9.87 J·K^-1·mol^-1, 吉布斯自由能变(ΔG)分别为20.61 kJ·mol^-1和20.49 kJ·mol^-1。     

The mechanism of tubulin-colchicine recognition--a kinetic study of the binding of a bicyclic colchicine analogue with a minor modification of the A ring

2-Methoxy-5-(2',3',4'-trimethoxy)-2,4,6-cycloheptatrien-1-one (MTC) is a colchicine analogue that lacks the B ring. 2-Methoxy-5-(2',4'-dimethoxyphenyl)-2,4,6-cycloheptatrien-1-one (MD) is an A-ring analogue of MTC, in which one methoxy group is replaced by a hydrogen atom. This paper describes the kinetic features of MDC binding to tubulin, and compares its behaviour with MTC to analyse the effect of the A-ring modification on the recognition process by tubulin. Binding is accompanied by a strong enhancement of MDC fluorescence and quenching of protein fluorescence. The kinetic and thermodynamic parameters were obtained from fluorescence stopped-flow measurements. The kinetics are described by a single exponential, indicating that this drug does not discriminate between the different tubulin isotypes. The observed pseudo-first-order rate constant of the fluorescence increase upon binding increases in a non-linear way, indicating that this ligand binds with a similar overall mechanism as colchicine and MTC, consisting of a fast initial binding of low affinity followed by a slower isomerisation step leading to full affinity. The K1 and k2 values for MDC at 25 degrees C were 540 +/- 65 M(-1) and 70 +/- 6 s(-1) respectively. From the temperature dependence, a reaction enthalpy change (deltaH(o)1) of the initial binding of 49 +/- 11 kJ/mol(-1) and an activation energy for the second step of 28 +/- 9 kJ/mol(-1) were calculated. Displacement experiments of bound MDC by MTC allowed the determination of a rate constant of reverse isomerisation of 0.60 +/- 0.07 s(-1) at 25 degrees C and the activation energy of 81 +/- 6 kJ/mol(-1). The overall binding constant was (6.3 +/- 0.2) x 10(4) M(-1) at 25 degrees C. Combination of these results with the kinetic parameters for association gives a full characterisation of the enthalpy pathway for the binding of MDC. The pathway of MDC is shown to differ considerably from that of MTC binding. Since its structural difference is located in ring A, this result indicates the use of ring A in the first step. The kinetics of the binding of MDC in the presence of some A-ring colchicine analogues (podophyllotoxin, 3',4',5'-trimethoxyacetophenone and N-acetylmescaline) and a C-ring analogue (tropolone methyl ether) suggest that the A and C rings are involved in the binding of MDC.     

Folding and conformational studies on SCR1-3 domains of human complement receptor 1

Short consensus repeats SCR3 and SCR1-3 are soluble recombinant proteins, consisting of the third and first three N-terminal domains of complement receptor 1, respectively, which retain some anti-complement activity. The conformational stabilities and folding/unfolding of SCR3 and SCR1-3 have been studied using circular dichroism and equilibrium and pre-equilibrium fluorescence spectroscopy. Denaturation by guanidinium hydrochloride (GdnHCl) is rapid and completely reversible. Reduction of disulphide bridges in the folded proteins by beta-mercaptoethanol leads to an increase in fluorescence intensity. The fluorescence intensity of the folded proteins is approximately 7.5% of that of the respective unfolded proteins. The data can be approximated to a two-state transition between native and denatured forms of the proteins. SCR3 has a conformational stability in water of 12-13 kJ/mol whereas that of SCR1-3 is 19.5-19.9 kJ/mol depending upon the technique utilized. The heat capacity change associated with the unfolding of SCR1-3 was obtained by a series of GdnHCl unfolding experiments over a range of temperatures and was found to be 6.6 kJ/K.mol or 33.8 J/K.mol(residue). The refolding process of SCR3 was found to be simple, described by a single exponential equation, whereas that of SCR1-3 was found to be complex and could be fitted to a double exponential equation indicating the presence of folding intermediates.     

Neutrophil-specific antigen NB1 inhibits neutrophil-endothelial cell interactions

Neutrophil-specific antigen NB1 is located on a 58 to 64 kd glycosyl phosphatidylinositol-linked plasma membrane glycoprotein. NB1 antigen can be detected on neutrophils from 97% of healthy volunteers, and NB1 antigen is expressed on subpopulations of neutrophils. Neutrophil subpopulations with varying functions have been described, and we hypothesize that NB1 antigen may play an important role in neutrophil function. We compared the function of NB1-positive and NB1-negative neutrophils obtained from several persons. There were no differences in the adhesion of NB1-positive and NB1-negative neutrophils incubated in C5a, N-formyl-Met-Leu-Phe (FMLP), phorbol myristate acetate (PMA), or buffer to type IV collagen, fibronectin, laminin, or polystyrene. However, the adherence to human umbilical vein endothelial cells (HUVEC) monolayers of unstimulated NB1-positive neutrophils was less than to NB1-negative neutrophils (20.0% +/- 4.2% vs 31.7% +/- 5.8%; p < 0.01). When neutrophils were stimulated with C5a, PMA, or FMLP, no differences were found in the adhesion of NB1-positive and NB1-negative cells to the same surfaces. When NB1-positive neutrophils were incubated with rabbit polyclonal anti-NB1 Fab fragments, their adherence to HUVEC was increased (32.9% +/- 10.1% vs 18.3% +/- 5.0%; p < 0.05). Fab fragments prepared from normal rabbit serum had no effect on neutrophil adherence to HUVEC. The chemotaxis of NB1-positive neutrophils to FMLP through nitrocellulose was significantly greater than that of NB1-negative neutrophils (p = 0.03), but there was no difference in chemotaxis to FMLP through polycarbonate membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermodynamics of oligoarginines binding to RNA and DNA

We have examined the equilibrium binding of a series of synthetic oligoarginines (net charge z = +2 to +6) containing tryptophan to poly(U), poly(A), poly(C), poly(I), and double-stranded (ds) DNA. Equilibrium association constants, K(obs), measured by monitoring tryptophan fluorescence quenching, were examined as functions of monovalent salt (MX) concentration and type, as well as temperature, from which deltaG(standard)obs, deltaH(obs), and deltaS(standard)obs were determined. For each peptide, K(obs) decreases with increasing [K+], and the magnitude of the dependence of K(obs) on [K+], delta log K(obs)/delta log[K+], increases with increasing net peptide charge. In fact, the values of delta log K(obs)/delta log[K+] are equivalent for oligolysines and oligoarginines possessing the same net positive charge. However, the values of K(obs) are systematically greater for oligoarginines binding to all polynucleotides, when compared to oligolysines with the same net charge. The origin of this difference is entirely enthalpic, with deltaH(obs), determined from van't Hoff analysis, being more exothermic for oligoarginine binding. The values of deltaH(obs) are also independent of [K+]; therefore, the salt concentration dependence of deltaG(standard)obs is entirely entropic in origin, reflecting the release of cations from the nucleic acid upon complex formation. These results suggest that hydrogen bonding of arginine to the phosphate backbone of the nucleic acids contributes to the increased stability of these complexes.     

Polyisoprenyl phosphates in intracellular signalling

In response to environmental stimuli, leukocyte membrane remodelling generates biologically active lipids that can serve as both intra- and extracellular mediators. There are several classes of lipids that can mediate inflammatory reactions. We report here on a new intracellular lipid signal that regulates oxygen-radical formation in neutrophils, a key response in microbial killing, inflammation and tissue injury. Screening of neutrophil-derived extracts rich in phosphorylated, non-saponifiable lipids revealed a potent inhibitor of superoxide anion (O2-) production. Structural analysis of biologically active fractions gave four major phosphorylated lipids: most abundant was presqualene diphosphate (PSDP). Upon activation of neutrophil receptors, PSDP and its monophosphate form, presqualene monophosphate (PSMP), undergo rapid remodelling. At submicromolar concentrations, PSDP but not PSMP inhibit O2- production by human neutrophil cell-free oxidase preparations. We prepared PSDP and PSMP by total organic synthesis and matched both the physical properties and biological activity of the neutrophil-derived compounds. Our results indicate that PSDP, a recognized intermediate of cholesterol biosynthesis, is present in immune effector cells and is a potent regulator of the cellular response in host defence.     

Evidence for the production of peroxynitrite in inflammatory CNS demyelination

Peroxynitrite, which is generated by the reaction of nitric oxide (NO) with superoxide, is a strong oxidant that can damage subcellular organelles, membranes and enzymes through its actions on proteins, lipids, and DNA, including the nitration of tyrosine residues of proteins. Detection of nitrotyrosine (NT) serves as a biochemical marker of peroxynitrite-induced damage. In the present studies, NT was detected by immunohistochemistry in CNS tissues from mice with acute experimental autoimmune encephalomyelitis (EAE). NT immunoreactivity was displayed by many mononuclear inflammatory cells, including CD4+ cells. It was also observed in astrocytes near EAE lesions. Immunostaining for the inducible isoform of NO synthase (iNOS) was also observed, particularly during acute EAE. These data strongly suggest that peroxynitrite formation is a major consequence of NO produced via iNOS, and implicate this powerful oxidant in the pathogenesis of EAE.     

曙红Y-藏红T能量转移荧光猝灭法测定痕量锰

在pH 6.0介质中,十二烷基苯磺酸钠(SDBS)作用下,曙红Y-藏红T能发生有效的能量转移,使藏红T荧光增强,二价锰的加入使藏红T在577nm处发生荧光猝灭,其荧光猝灭程度与锰的含量呈线性关系,由此建立了能量转移荧光测定痕量锰的新方法。试验探讨了体系的最佳条件:1.0×10^-6 mol/L曙红Y溶液0.5mL、1.0×10^-5 mol/L藏红T溶液4.0mL、1.0×10^-3 mol/L SDBS溶液0.3mL,40℃恒温水浴中水浴加热4min。在优化条件下,锰(Ⅱ)质量浓度在0.4×10^-8~6.0×10^-8 mol/L范围内与荧光猝灭程度呈线性响应,方法检出限为6.0×10^-9 mol/L。将该体系应用于茶叶和土壤样品中锰的测定,相对标准偏差(RSD,n=11)小于5%,回收率为93.8%~95.6%。     

Effects of temperature on the kinetics of the gated electron-transfer reaction between zinc cytochrome c and plastocyanin. Analysis of configurational fluctuation of the diprotein complex

This is a study of the effects of temperature (in the range 273.3-307.7 K) and of ionic strength (in the range 2.5-100 mM) on the kinetics of photoinduced electron-transfer reaction 3Zncyt/pc(II)--> Zncyt+/pc(I) within the electrostatic complex of zinc cytochrome c and cupriplastocyanin at pH 7.0. In order to separate direct and indirect effects of temperature on the rate constants, viscosity of the solutions was fixed, at different values, by additions of sucrose. The activation parameters for the reaction within the preformed complex, at the low ionic strength, are delta H++ = 13 +/- 2 kJ/mol and delta S++ = -97 +/- 4 J/K mol. The activation parameters for the reaction within the encounter complex, at the higher ionic strength, are delta H++ = 13 +/- 1 kJ/mol and delta S++ = -96 +/- 3 J/K mol. Evidently, the two complexes are the same. The proteins associate similarly in the persistent and the transient complex, i.e., at different ionic strengths. In both complexes, however, electron transfer is gated by a rearrangement, as previous studies from this laboratory showed. Changes in the solution viscosity modulate this rearrangement by affecting delta H++, not delta S++. The activation parameters are analyzed by empirical methods. The thermodynamic parameters delta H and delta S for the formation of the complex Zncyt/pc(II) are determined and related to changes in hydrophilic and hydrophobic surfaces upon protein association in three configurations. A difference between the values of delta H for the configuration providing optimal electronic coupling between the redox sites and the configuration providing optimal docking equals the experimental value delta H++ = 13 kJ/mol for the rearrangement of the latter configuration into the former. Enthalpy of activation may reflect a change in the character of the exposed surface as the diprotein complex rearranges. Entropy of activation may reflect tightening of the contact between the associated proteins.     

Involvement of protein kinase C and of protein phosphatases 1 and/or 2A in p47 phox phosphorylation in formylmet-Leu-Phe stimulated neutrophils: studies with selective inhibitors RO 31-8220 and calyculin A

Previously employed non-selective protein kinase inhibitors yielded inconclusive results regarding involvement of protein kinase C (PKC) in phosphorylation of 47 kDa protein (p47 phox) in intact neutrophils stimulated with physiologic agonists of superoxide generation. In the present study, phosphorylation of p47 phox in formylMet-Leu-Phe (fMLP) stimulated neutrophils was potently inhibited in the presence of 0.3 microM RO 31-8220, a selective inhibitor of PKC. These results provide experimental evidence in support of the currently considered essential involvement of PKC in p47 phox phosphorylation in response to physiologic stimulation of neutrophil surface receptors. The fMLP-induced phosphorylation of p47 phox was enhanced and prolonged by calyculin A, a specific inhibitor of protein phosphatases of types 1 and 2A, and such enhanced phosphorylation was also effectively inhibited by RO 31-8220. Our results suggest that the extent and duration of p47 phox phosphorylation in intact fMLP-stimulated neutrophils is probably controlled by a balance between the activities of PKC, on the one hand, and of protein phosphatase(s) of type(s) 1 and/or 2A, on the other. Effects of RO 31-8220 and of calyculin A on the fMLP-induced p47 phox phosphorylation were paralleled by similar effects on superoxide release. Calyculin A and RO 31-8220 were also used to study signal transduction by a post-receptor agonist of superoxide generation, a calcium ionophore A23187. The results of the latter study indicated that PKC was activated in A23187-stimulated neutrophils and was essentially involved in superoxide generation and p47 phox phosphorylation. Further, these results suggested that protein phosphatase(s) of type(s) 1 and/or 2A were also activated in A23187-signalling pathway, and limited the extent of superoxide release and p47 phox phosphorylation.     

Role of phosphatidylethanolamine lipids in the stabilization of protein-lipid contacts

We have investigated the effect of lipids with phosphatidylethanolamine (PE) head groups on the stabilization of contacts between the tryptophan side chains of gramicidin and the lipid head groups. We initially developed two fluorescence methods that can be correlated to the spontaneous curvature of DOPC/DOPE and DOPC/DOPEme. One is based on bilayer structure and measures the rotational motion of a probe located close to the membrane surface relative to a more deeply-buried probe. The second is based on surface hydration/polarity and measures the emission energy of a polarity-sensitive probe located on the membrane surface. We used these methods to estimate the pseudo-curvature (i.e., curvature obtained by fluorescence measurements) of lipids with dimyristyl chains, and their pressure and temperature dependence. We then investigated the stability of gramicidin tryptophan-lipid contacts in DMPC/DMPE as a function of temperature and pressure. Stability was assessed by tryptophan rotational motion as determined by fluorescence anisotropy, since rotational motion is limited when the indoles are hydrogen bonded to the lipid head groups. The results suggest that the presence of PE lipids destabilizes these contacts due to either their smaller size relative to PC head groups, or their tendency to self-interact. Fluorescence quenching studies support these results.     

地西他滨联合三氧化二砷对NB4细胞凋亡作用的研究

目的 研究去甲基化制剂地西他滨(DAC)单用或联合三氧化二砷(As2O3)对NB4细胞凋亡的作用机制.方法 将不同浓度的DAC、As2O3以及两药联合作用于NB4细胞,不加药为对照组,采用四甲基偶氮唑蓝(MTT)法检测细胞增殖抑制作用,流式细胞术检测细胞凋亡.结果 DAC与As2O3单药对NB4细胞的抑制作用呈浓度时间依赖性(DAC 1 μmol/L作用24、48、72 h的抑制率分别为12.18%、22.72%、35.54%;DAC 2 μmol/L作用24、48、72 h的抑制率分别增高为22.14%、31.18%、45.21%;As2O3 0.5 μmol/L作用24、48、72 h的抑制率分别21.09%、32.43%、44.93%;As2O3 1.0 μmol/L作用24、48、72 h的抑制率分别增高为31.69%、41.12%及54.27%),两药联合抑制作用较单药明显(DAC 1 μmol/L+As2O3 0.5 μmol/L作用24、48、72 h抑制率分别为42.10%、48.75%、60.78%)(P<0.05),各浓度组与对照组比较差异均有统计学意义(P值均<0.05);As2O3 1 μmol/L作用于NB4细胞株48 h可见5.8%的细胞凋亡,联合组增高为17.3%.结论 DAC能显著抑制NB4细胞的增殖并诱导其凋亡,DAC联合As2O3对NB4细胞增殖抑制及诱导凋亡有协同作用.     

Differential superoxide anion generation by equine eosinophils and neutrophils

Equine eosinophils and neutrophils are believed to play an important part in the protection of horses against parasitic and bacterial invasion. Eosinophils may also play a key role in the pathogenesis of equine inflammatory conditions such as the allergic skin disease, insect hypersensitivity. The factors which stimulate the respiratory burst of equine eosinophils and neutrophils are poorly understood. The first aim of the present study was to determine the effects of the phorbol ester, phorbol myristate acetate (PMA), which is believed to activate intracellular protein kinase C, and opsonised particles of serum-treated zymosan (STZ), on the production of superoxide anions by equine eosinophils and neutrophils. Since histamine has been detected after antigen challenge in the skin of horses with insect hypersensitivity, the second aim was to establish the effects of this mediator on superoxide anion production by equine eosinophils and the receptor sub-type(s) that mediate histamine-induced responses. For comparison, responses of neutrophils from the same horses were also examined. PMA and STZ induced significant increases in superoxide anion generation by equine eosinophils and neutrophils. The estimated maximum (EMAX) superoxide anion production by eosinophils in the presence of PMA was significantly greater than that of neutrophils; the estimated concentration of PMA inducing 50% of the maximum response (EC50) by eosinophils was significantly less. The EMAX values for superoxide anion production by neutrophils in the presence of STZ were significantly greater than those for eosinophils. Histamine induced superoxide anion generation by equine eosinophils which was inhibited by the histamine-1 receptor antagonists chlorpheniramine and mepyramine, but not the histamine-2 and histamine-3 receptor antagonists, cimetidine and thioperamide, respectively. Histamine did not cause superoxide anion production by equine neutrophils. These studies demonstrate that equine granulocytes vary in their ability to produce a respiratory burst in the presence of different stimuli, with eosinophils being more responsive to protein kinase C activators and neutrophils to opsonised particles. They also show that histamine selectively induced the generation of superoxide anions by equine eosinophils via histamine-1 receptor activation. Thus, in horses with insect hypersensitivity, histamine released from cutaneous mast cells after antigen challenge could activate eosinophils which have migrated into the dermis.     

Effect of aromatic nitroso-compounds on superoxide-generating activity in neutrophils

Aromatic nitroso-compounds such as nitrosobenzene inhibited the respiratory burst of intact neutrophils induced by various stimulants, including phorbol 12-myristate 13-acetate and a chemotactic peptide. The compounds also inhibited NADPH-dependent oxygen consumption by cell-free preparations of neutrophils. This indicates that nitroso-compounds act directly on the NADPH-oxidase system. The inhibitory effects induced by several nitroso-compounds, 2-nitrosotoluene, nitrosobenzene, 4-nitrosophenol, and 1-nitrosopyrrolidine, were examined and their inhibition constants, the concentrations causing 50% reduction of oxygen consumption, were found to be 0.043, 0.173, 0.672, and 32.1 mM, respectively. These values correlated well with the hydrophobicity of the compounds: a more hydrophobic compound was a more potent inhibitor against NADPH oxidase, suggesting that the oxidase has a hydrophobic site(s) for interaction with the inhibitors.