Optical probes for detection and quantification of neutrophils’ oxidative burst. A review

Neutrophils, also known as polymorphonuclear leukocytes (PMN), are the most common type of white blood cells, comprising about 50-70% of all white blood cells. In the event of inflammatory processes, neutrophils display increased mobility, tissue influx ability, prolonged life span, and an increased phagocytic capacity, constituting the initial participants in the cellular defense of the organism. One of the most important defense systems of neutrophils corresponds to their ability to mediate a strong oxidative burst through the formation of reactive oxygen species (ROS) and reactive nitrogen species (RNS). While oxidative burst is important for the elimination of invading microorganisms, the overproduction of ROS and RNS or the impairment of endogenous antioxidant defenses may result to detrimental effects to the host. The nature and the extent of ROS and RNS production by neutrophils in response to different stimuli is, consequently, a matter of extensive research, with scientific reports showing an enormous variability on the detection methodologies employed. This review attempts to provide a critical assessment of the most common approaches to identify and quantify reactive species formed during the neutrophils’ oxidative burst. The detection mechanisms and performance, as well as advantages and limitations of the different methodologies, are scrutinized, focusing on the use of fluorimetric, chemiluminometric and colorimetric probes.     

Quantitative analysis of human serum leptin using a nanoarray protein chip based on single-molecule sandwich immunoassay

We report a method for the quantitative analysis of human serum leptin, which is a protein hormone associated with obesity, using a nanoarray protein chip based on a single-molecule sandwich immunoassay. The nanoarray patterning of a biotin-probe with a spot diameter of 150 nm on a self-assembled monolayer functionalized by MPTMS on a glass substrate was successfully accomplished using atomic force microscopy (AFM)-based dip-pen nanolithography (DPN). Unlabeled leptin protein molecules in human serum were detected based on the sandwich fluorescence immunoassay by total internal reflection fluorescence microscopy (TIRFM). The linear regression equation for leptin in the range of 100 zM-400 aM was determined to be y = 456.35x + 80,382 (R = 0.9901). The accuracy and sensitivity of the chip assay were clinically validated by comparing the leptin level in adult serum obtained by this method with those measured using the enzyme-linked immunosorbent assay (ELISA) performed with the same leptin standards and serum samples. In contrast to conventional ELISA techniques, the proposed chip methodology exhibited the advantages of ultra-sensitivity, a smaller sample volume and faster analysis time.     

规模化蛋白质生物质谱鉴定中肽段氨基端环化修饰现象

对蛋白质样品制备中引入的氨基酸残基的一种现象--蛋白质酶切肽段氨基端的环化修饰现象的初步研究结果显示,很多以谷氨酰胺(Q)或氨乙酰化修饰的半胱氨酸(CAM_C)残基起始的肽段会发生氨基端的环化修饰,且修饰反应不完全,在同一样本中修饰与非修饰两种状态常同时存在,并且环化修饰后的肽段的反相色谱保留时间发生延迟.在数据库检索时添加环化修饰,可以提高蛋白质的鉴定成功率.本研究结果为大规模的蛋白质质谱数据解析提供了有价值的参考.     

Specific interaction of jacalin with phycocyanin, a fluorescent phycobiliprotein

Recent research has shown that, like porphyrins, phycocyanin (PC) too can produce singlet oxygen upon excitation with the appropriate radiation and hence could be useful in photodynamic therapy (PDT) for cancer. Unlike porphyrins, PC has the advantage of being a non-toxic, non-carcinogenic, soluble protein. However, the challenge would be to target the fluorescent phycobiliprotein to malignant cells. We report here that the tumor-specific lectin, jacalin, binds PC specifically in a carbohydrate-independent manner and with affinities better than that for porphyrins. Hence the lectin could prove to be a useful carrier for targeted delivery of PC. The interaction involves both ionic and hydrophobic interactions and more than one contact site.     

基于串级质谱信息进行蛋白质数据库搜索的结果可靠性分析

SEQUEST与Mascot为目前蛋白组学分析研究中使用最为广泛的蛋白质库搜索工具.尝试将Mascot与SEQUEST搜索结果进行比较,进而采用不同多变量判别方法对二者的搜索结果进行判别分析,以降低其结果的假阳性率.通过对Mascot与SEQUEST搜索结果进行比较,发现所得结果差异很大;利用多变量判别分析方法对Mascot及SEQUEST搜索结果进行判别分析,可有效提高SEQUEST结果中假阳性结果与正确结果之间的区分能力.对于Mascot搜索结果,采用多变量判别分析方法仍无法显著降低其假阳性结果,利用Decoy库搜索结果进行估计时亦存在导致错误估计的风险.     

Ordered nano-structure of a stamped self-organized protein layer on a HOPG surface using a HFB carrier

A groundbreaking method for ordered molecular layer preparation on a solid surface employing the drop-stamp method has been developed by us taking advantage of the characteristics of the HFB molecule as a self-organizer/adsorption carrier. It is a smart method which can be used to prepare a self-organized protein layer on a solid surface without unspecific adsorption or defects. In our previous report, we clarified the self-organizing nature of HFB-tagged protein molecules on a surface of a solution droplet. In this report, a protein layer was prepared on a HOPG surface by using the drop-stamp method with a maltose binding protein (MBP)-tagged HFBII molecule. The structure of the stamped protein layer was investigated using frequency modulation atomic force microscopy (FM-AFM) in a liquid condition. The FM-AFM images show that the drop-stamp method can prepare an ordered protein layer on a solid surface smartly. The drop-stamp method using a HFB carrier is a practical method which can be used to prepare an ordered protein layer on a solid substrate surface without unspecific adsorption defects.     

An Optimised Small‐Molecule Stabiliser of the 14‐3‐3–PMA2 Protein–Protein Interaction

Modulation of protein–protein interactions (PPIs) is a highly demanding, but also a very promising approach in chemical biology and targeted drug discovery. In contrast to inhibiting PPIs with small, chemically tractable molecules, stabilisation of these interactions can only be achieved with complex natural products, like rapamycin, FK506, taxol, forskolin, brefeldin and fusicoccin. Fusicoccin stabilises the activatory complex of the plant H⁺‐ATPase PMA2 and 14‐3‐3 proteins. Recently, we have shown that the stabilising effect of fusicoccin could be mimicked by a trisubstituted pyrrolinone (pyrrolidone1, 1 ). Here, we report the synthesis, functional activity and crystal structure of derivatives of 1 that stabilise the 14‐3‐3–PMA2 complex. With a limited compound collection three modifications that are important for activity enhancement could be determined: 1) conversion of the pyrrolinone scaffold into a pyrazole, 2) introduction of a tetrazole moiety to the phenyl ring that contacts PMA2, and 3) addition of a bromine to the phenyl ring that exclusively contacts the 14‐3‐3 protein. The crystal structure of a pyrazole derivative of 1 in complex with 14‐3‐3 and PMA2 revealed that the more rigid core of this molecule positions the stabiliser deeper into the rim of the interface, enlarging especially the contact surface to PMA2. Combination of the aforementioned features gave rise to a molecule ( 37 ) that displays a threefold increase in stabilising the 14‐3‐3–PMA2 complex over 1 . Compound 37 and the other active derivatives show no effect on two other important 14‐3‐3 protein–protein interactions, that is, with CRaf and p53. This is the first study that describes the successful optimisation of a PPI stabiliser identified by screening.     

Multivalent Design of Apoptosis‐Inducing Bid‐BH3 Peptide–Oligosaccharides Boosts the Intracellular Activity at Identical Overall Peptide Concentrations

Multivalent peptide–oligosaccharide conjugates were prepared and used to investigate the multivalency effect concerning the activity of Bid‐BH3 peptides in live cells. Dextran oligosaccharides were carboxyethylated selectively in the 2‐position of the carbohydrate units and activated for the ligation of N‐terminally cysteinylated peptides. Ligation through maleimide coupling was found to be superior to the native chemical ligation protocol. Monomeric Bid‐BH3 peptides were virtually inactive, whereas pentameric peptide conjugates induced apoptosis up to 20‐fold stronger at identical peptide concentrations. Comparison of lowly multivalent and highly multivalent peptide dextrans proved a multivalency effect in life cells which was specific for the BH3 peptide sequence.