Proteomic analysis of the response of the human neutrophil-like cell line NB-4 after exposure to anthrax lethal toxin

We used 2‐D DIGE to analyze the early response of NB‐4 cells, a human promyelotic leukemia cell line, exposed to lethal toxin from Bacillus anthracis at the proteome level. After a 2 h exposure, cells were still viable and 43% of spots (n = 1042) showed a significant change in protein level. We identified 59 spots whose expression had changed significantly, and these reflected cytoskeleton damage, mitochondrial lysis and endoplasmic reticulum stress. Actin filament assembly was disrupted as evidenced by an increase in both actin subunits and phosphorylated cofilin, whilst levels of tropomyosin, tropomodulin and actin related protein 2/3 complex subunit decreased. Lower levels of ATP synthase subunits and mitochondrial inner membrane protein were identified as markers of mitochondrial lysis. Levels of various stress response proteins rose and, uniquely, levels of Ca²⁺ binding proteins such as translationally controlled tumor protein rose and hippocalcin‐like protein 1 decreased. This response may have mitigated effects brought about by mitochondrial lysis and endoplasmic reticulum stress, and delayed or prevented apoptosis in NB‐4 cells. These results resemble findings of similar proteomics studies in murine macrophages, although quantitative differences were observed.     

Rotary shadowing with platinum-carbon in biological electron microscopy: A review of methods and applications

Rotary shadowing when combined with such specimen preparation techniques as quick freezing and deep etching, critical point drying, and glycerol spraying is a highly versatile method of visualizing cell and macromolecular ultrastructure. This review outlines the procedures commonly used to prepare specimens for rotary shadowing and evaluates the relative merits of rotary shadowing when compared to unidirectional shadowing and negative staining.     

Isolation of saponin-free fraction from Ginseng (Panax ginseng C.A. Meyer) and its effects on the function of neutrophils

A method of isolation of bioactive water-soluble saponin-free subfraction (RG-ws-1-1-1) from Korean red ginseng using solvent extraction, ultrafiltration and chromatography on Diaion HP-20 resin was developed. To evaluate the potency of RG-ws-1-1-1 to effect on the heat stress (HS) response and anti-inflammatory response in neutrophils, the oxygen consumption (OC) and formation of Superoxide radicals (FSR) were assayed in suspensions of neutrophils from mice. Supplementation of RG-ws-1-1-1 to micein vivo (200 mg/kg, 14 days intraperitoneally) results in the increase OC by neutrophils assayedex vivo at 37 ℴC. OC and FSR by neutrophils were suppressed when cells obtained from the control animals were incubated at 42 ℴC. Preincubation of neutrophils with RG-ws-1-1-1 reduces HS related depression of FSR. Supplementation of RG-ws-1-1-1 to micein vivo decreases HS related suppression of OC. RG-ws-1-1-1 subfraction significantly improves an oxygen-dependent anti-microbial function of neutrophils, also studied under the HS conditions.     

Platelet-Activating Factor Quantification Using Reversed Phase Liquid Chromatography and Selected Reaction Monitoring in Negative Ion Mode

Platelet‐activating factor (PAF) is a potent biologically active phospholipid that mediates human physiological and pathophysiologic responses. PAF levels increase transiently and are typically assessed by techniques with limitations related to expense, sensitivity, pre‐analysis derivatization and interference with isobaric molecules. This study elucidates a facile, accurate liquid chromatography–mass spectrometry analytical method for PAF. In negative ion mode using electrospray ionization, collisionally‐activated dissociation analysis showed a unique product ion for acetate adducts of PAF molecular species representing the loss of methyl acetate from the polar head group and loss of a part of the acetate group from the sn‐2 position. This product ion was exploited for selected reaction monitoring of PAF molecular species following separation by reversed‐phase liquid chromatography. Standard calibration responses were determined, and this method was able to detect as low as 100 fmol of PAF. Finally, PAF molecular species were quantified in human neutrophils and monocytes.     

Evaluation of the antioxidant activity of passion fruit (Passiflora edulis and Passiflora alata) extracts on stimulated neutrophils and myeloperoxidase activity assays

The antioxidant activity of methanol extracts from Passiflora edulis and Passiflora alata pulp, and P. edulis rinds, healthy or infected with the passion fruit woodiness virus (PWV), was investigated using the oxidant activities of the neutrophil and the neutrophil granule enzyme myeloperoxidase (MPO), both playing key roles in inflammation. The reactive oxygen species produced by stimulated neutrophils were evaluated by lucigenin-enhanced chemiluminescence (CL) and the activity of purified MPO was measured by SIEFED (Specific Immunological Extraction Followed by Enzymatic Detection), a technique for studying the direct interaction of a compound with the enzyme. The rind extracts of P. edulis possessed higher and dose-dependent inhibitory effects on CL response and on the peroxidase activity of MPO than total pulp extracts from both passion fruit species. The quantification of isoorientin in the extracts showed a correlation with their antioxidant activity, suggesting the potential of P. edulis rinds as functional food or as a possible source of natural flavonoids.     

肺炎链球菌表面蛋白C对人嗜中性粒细胞释放CXCL8的影响

目的原核表达肺炎链球菌表面蛋白C(Pneumococcal surface protein C,PspC),并分析该蛋白对人嗜中性粒细胞释放CXCL8的影响。方法将重组质粒pET-32a(+)/PspC转化至大肠杆菌BL21(DE3)中,经IPTG诱导表达TRx-His-PspC融合蛋白并纯化;通过肠激酶切掉融合蛋白的TRx-His部分,获得PspC蛋白,免疫小鼠,获得抗PspC抗体。分离人外周血嗜中性粒细胞,经MTT法检测PspC蛋白对嗜中性粒细胞增殖活力的影响;QRT-PCR检测PspC蛋白及抗PspC抗体对人嗜中性粒细胞释放CXCL8的影响。结果表达的TRx-His-PspC融合蛋白相对分子质量约为80 000,目的蛋白约占菌体总蛋白的21%,可与His Tag McAb特异性结合。获得的PspC蛋白相对分子量约为60 000,免疫小鼠获得了高滴度的抗PspC中和抗体(1∶12 000)。分离的人外周血嗜中性粒细胞纯度>98%,活细胞比例大于96%,经不同浓度的PspC蛋白刺激人嗜中性粒细胞,不会引起宿主细胞的细胞毒作用;不同浓度的PspC蛋白刺激人嗜中性粒细胞8 h后,CXCL8基因mRNA转录水平显著上调(P<0.01),且呈剂量依赖性;CXCL8的释放均呈剂量依赖性增强(P<0.01),且24 h高于12 h;抗PspC抗体可显著抑制PspC蛋白刺激人嗜中性粒细胞释放CXCL8。结论 PspC蛋白可上调人嗜中性粒细胞趋化因子CXCL8的合成和释放,揭示了嗜中性粒细胞和肺炎链球菌致病因素之间的关系,为控制肺炎链球菌侵入性疾病奠定了基础。     

Antioxidant and anti-inflammatory activities of Ribes nigrum extracts

Blackcurrant berries contain high amounts of flavonoids with various health benefits as anti-inflammatory properties attributed to their antioxidant potential. Leaves and buds actually used to produce food supplement could also exhibit such interesting properties.In the literature, various methods are often used and valid indicators of the antioxidant potential of dietary substances. However these assays do not provide evidence that antioxidants have in vivo or ex vivo activity when consumed. To obtain biologically relevant information, the antioxidant activities of the extracts were evaluated on cellular models implicating the measurement of blood haemolysis, the Cellular Antioxidant Activity on endothelial cells and the anti-inflammatory activities on isolated equine stimulated neutrophils and purified myeloperoxidase.These tests generally showed that the blackcurrant leaf extract have the highest antioxidant and anti-inflammatory (inhibition of MPO activity and ROS production on activated neutrophils) capacities correlated to the highest total phenolics content.