Auranofin affects early events in human polymorphonuclear neutrophil activation by receptor-mediated stimuli

Auranofin, a new oral antirheumatic gold compound, in concentrations achieved therapeutically, inhibits neutrophil phagocytosis, chemotaxis, chemiluminescence, reduction of cytochrome c, and release of lysosomal enzymes. To further characterize the mechanism by which auranofin affects neutrophils, we studied the effects of auranofin on unstimulated properties and functions of neutrophils as well as on rapidly stimulated functions. When examined by electron microscopy, 4 micrograms/ml of auranofin significantly decreased the number of visualized centriole-associated microtubules in resting cells. Furthermore, auranofin inhibited neutrophil spreading on glass and caused a decrease in negative surface charge (electrophoretic mobility). In addition, auranofin inhibited several fmet-leu-phe-stimulated responses such as shape change, increases in centriole-associated microtubules, decreases in surface charge, and elicited membrane potential changes (di-O-C5(3) dye response). Auranofin (1 micrograms/ml) inhibited fmet-leu-phe-stimulated superoxide and hydrogen peroxide production by 80% (p less than 0.05), and also increased the affinity of receptors for fmet-leu-phe (from Ka 0.035 to Ka 0.48, p less than 0.001). Auranofin also affected neutrophil responses to phorbol myristic acetate (PMA). The total amount of PMA-stimulated superoxide production was suppressed by as little as 0.4 micrograms/ml of auranofin, but the lag time for activation was shortened by low concentrations of auranofin (0.5 to 1 microgram/ml). Four micrograms per milliliter of auranofin suppressed the decrease in surface charge induced by PMA. However, auranofin did not influence superoxide production elicited by the ionophore A23187. The results indicate that auranofin affects the earliest detected responses in neutrophil activation by certain receptor-mediated stimuli.     

Intravenous endotoxin recruits a distinct subset of human neutrophils, defined by monoclonal antibody 31D8, from bone marrow to the peripheral circulation

Human neutrophils label with fluorochrome-labeled monoclonal antibody 31D8 as bright or dull. We determined the source and fate of 31D8 dull neutrophils by studying volunteers injected with endotoxin, epinephrine, or hydrocortisone, by examining bone marrow, and by examining skin blister exudate. We find that 31D8 dull neutrophils are normally not present in significant numbers in the circulation, are present in large numbers in normal marrow, and are recruited from the marrow by endotoxin, to a lesser extent by steroid, but not at all by epinephrine. 31D8 dull pattern correlates with morphologic immaturity in postendotoxin peripheral blood and bone marrow; however, blister exudate neutrophils contain only morphologically mature neutrophils, of which a significant number are 31D8 dull. We conclude that 31D8 dull neutrophils reside primarily in bone marrow and are released by agents which enhance bone marrow release of neutrophils. Their accumulation in skin blister exudate is unexplained, but suggests a special role in the inflammatory process.     

Subcellular localization of Gi alpha in human neutrophils

Subcellular fractions were prepared from human neutrophils by sucrose density gradient centrifugation and analyzed for Gi-like proteins by pertussis toxin-catalyzed [32P]ADP-ribosylation and by immunoblotting with rabbit antiserum AS/6 which recognizes purified transducin and Gi, but not Gs or Go alpha-subunits. In resting cells, approximately equal to 60% of pertussis toxin substrate retrieved from the sucrose density gradient localized to the plasma membrane-enriched fraction, approximately equal to 35% to the specific granule-enriched fraction, and approximately equal to 5% to cytosol. The azurophil granule-enriched fraction did not contain pertussis toxin substrate. In contrast to plasma membrane, the specific granule-enriched fraction demonstrated increased AS/6 immunoreactivity of a approximately equal to 41-kDa protein relative to a approximately equal to 40-kDa protein. Within the specific granule-enriched fraction, the peak of pertussis toxin substrate detected immunochemically or by [32P]ADP-ribosylation sedimented at a lighter density (rho = 1.6 g/ml) than did lactoferrin (rho = 1.19 g/ml), suggesting that the intracellular compartment bearing pertussis toxin substrate may not be the lactoferrin containing specific granule, per se. Furthermore, in neutrophils exposed to 10(-8) M N-formylmethionylleucylphenylalanine, a weak degranulating stimulus (7% lactoferrin degranulation), there was a 31-42% decline in pertussus toxin-catalyzed [32P]ADP-ribosylation of approximately equal to 40-41-kDa proteins in the specific granule-enriched fraction accompanied by a near-quantitative increase in labeling of plasma membrane. The pool of intracellular formyl peptide receptors localized to the specific granule-enriched fraction appeared functionally coupled to a cosedimenting G-protein in experiments demonstrating modulation of high affinity N-formylmethionylleucyl[3H]phenylalanine binding by guanosine 5'-(3-O-thio)triphosphate or pertussis toxin. The data indicate that neutrophils contain a surface translocatable pool of intracellular G-protein sedimenting in the specific granule-enriched fraction and support the view that mobilization of intracellular G-protein represents a mechanism by which cells can regulate receptor activity.     

Diffusion of extracellular hydrogen peroxide into intracellular compartments of human neutrophils. Studies utilizing the inactivation of myeloperoxidase by hydrogen peroxide and azide

It is well known that catalase is transformed to nitric oxide-Fe2+-catalase by hydrogen peroxide (H2O2) plus azide. In this report, we show that myeloperoxidase is also inactivated by H2O2 plus azide. Utilizing this system, we studied the presence and source of intracellular H2O2 generated by activated neutrophils. Stimulation of neutrophils with phorbol myristate acetate (PMA, 100 ng/ml) plus azide (5 mM) for 30 min completely inactivated intragranular myeloperoxidase and reduced cytosolic catalase to 35% of resting cells. This intracellular inactivation of heme enzymes did not occur in normal neutrophils incubated with either PMA or azide alone or in neutrophils from patients with chronic granulomatous disease (CDG) which cannot produce H2O2 in response to PMA. Incubation of neutrophils with azide and a H2O2 generating system (glucose-glucose oxidase) inactivated 41% of neutrophil myeloperoxidase. Glutathione-glutathione peroxidase (GSH-GSH peroxidase), an extracellular H2O2 scavenger, totally protected neutrophil myeloperoxidase from inactivation by azide plus glucose-glucose oxidase. In addition, when a mixture of normal and CGD cells was stimulated with PMA in the presence of azide, 90% of the myeloperoxidase in CGD neutrophils was inactivated. Therefore, H2O2 released extracellularly from activated neutrophils can diffuse into cells. In contrast, myeloperoxidase in normal polymorphonuclear leukocytes stimulated with PMA in the presence of azide and GSH-GSH peroxidase was 75% inactivated. Thus, the results indicate that a GSH-GSH peroxidase-insensitive pool of H2O2 is also generated, presumably at the plasma membrane, and this pool of H2O2 can undergo direct internal diffusion to inactivate myeloperoxidase.     

Nucleotide action on spontaneous electrical activity of calcium deficient nerve

In concentrations having no effect on the evoked alpha-A fiber spike, adenosine triphosphate (ATP), adenosine monophosphate (AMP) and several other nucleotides produced antagonism of spontaneous impulses in isolated desheathed frog nerve soaked in Ca free solution. ATP was only slightly more potent than AMP, indicating that high-energy phosphate bonds and Ca complexing are not important for stabilizing action. Furthermore, sub-effective concentrations of Ca potentiated the stabilizing action of ATP to a minimal degree and that of AMP not at all, suggesting a direct action of the nucleotide per se rather than a Ca-nucleotide complex. Ca⁴⁵ washout experiments showed that the nucleotides did not depress efflux of Ca from nerve axons but, in fact, caused release of Ca. It was proposed that nucleotide stabilization is associated with replacement of nucleotide lost from the excitable membrane into the Ca free medium.     

镉引起小麦苗逆境乙烯的产生及其和镉的吸收分布的关系

溶液培养小麦幼苗转移至含Cd~(2 )的营养液中,根系乙烯产生较快地增加,约在12h达高峰,然后下降;ACC含量亦呈先升后降的趋势。未和Cd~(2 )溶液直接接触的地上部乙烯亦增加,至36h达高峰,此后急剧下降,而ACG和 MAGC含量持续上升。地上部乙烯的增加,主要是由通过根系运往地上部的镉直接作用的结果,不是根部合成ACG运往地上部后再产生的。电镜观察表明,地上部乙烯产生和ACC含量变化的时间进程,可以与镉进入叶细胞内的部位及其对细胞膜和细胞器的影响相联系。     

评论：塑料结合模块促进聚对苯二甲酸乙二醇酯的酶法降解

塑料的大量生产和无节制的使用已造成严重的环境污染。为了减少塑料废物对环境的影响,近年来塑料酶法降解已成为国内外研究者关注的热点。例如,通过蛋白质工程策略提高塑料降解酶催化活性和热稳定性,进一步提高酶法降解的效率。另外,通过融合酶策略将塑料结合模块与塑料降解酶融合,也可以促进塑料降解。近期发表在期刊Chem Catalysis的一项研究表明,采用碳水化合物结合模块融合策略可以在低浓度(<10 wt%)的底物聚对苯二甲酸乙二醇酯[poly(ethylene terephthalate),PET]中提高塑料降解酶的活性。但是在高浓度底物(10 wt%−20 wt%)中,该策略无法提高PET的酶法降解。该项研究对于采用塑料结合模块促进酶法降解塑料具有重要的指导意义。     

ε-聚赖氨酸抑菌性能的研究进展

ε-聚赖氨酸(ε-poly-L-lysine,ε-PL)是抑菌谱广泛的天然抑菌剂,由通过α-羧基与ε-氨基连接的25–35个赖氨酸聚合而成。ε-PL主要由白色链霉菌发酵生产所得,比化学生产更加高效和环保。ε-PL具有水溶性好、耐热和对环境无污染等特点,具有良好的应用前景。本文从发酵生产入手,着重综述了ε-PL对各种微生物抑菌性能、抑菌机制及抑菌机制模型的研究进展。推测ε-PL是通过对细胞膜的破坏而改变细胞的通透性,或者作用到细胞内引起活性氧(reactive oxygen species, ROS)胁迫而影响调节基因的表达,从而起到抑菌作用。根据这2种抑菌方式分别建立了相应的抑菌模型,即毡毯模型和ROS诱导细胞凋亡模型。本文可为ε-PL对微生物抑制性能的深入研究提供依据,同时也提出了ε-PL抑菌机制的新模型,为扩展ε-PL应用领域提供了一定的参考。