Detection of brassinosteroids in pollen of Lolium perenne L. by immunocytochemistry

Bioactive brassinosteroids have been localized in developing and mature pollen of anhydrously fixed rye-grass (Lolium perenne) by immunocytochemistry using polyclonal antibodies to castasterone generated in rabbits. Tricellular pollen fixed by freeze-substitution was also labelled in the starch granules. Study of the developmental sequence of the pollen through the microsporocyte, microspore, bicellular and tricellular stages showed that the brassinosteroids were increasingly sequestered in starch granules as the amyloplasts matured, supporting the view that these are storage organelles for these potent plant growth promoters. In bicellular pollen, heavy labelling was seen in the zone within 0.5 μm of the starch granule, where stromal tissue remains. Thus, the stroma may be the site of synthesis of these compounds. During aqueous fixation, the brassinosteroids leached from the starch granules of tricellular pollen, indicating that they would be quickly available after imbibition to influence the physiology of germinating pollen. The results from high-performance liquid chromatography of dansylaminophenylboronates from partially purified extracts of freshly dehisced tricellular pollen of rye-grass showed 25-methylcastasterone may be a minor component, together with two unknown peaks. No specific binding of brassinolide to any soluble proteins extracted from tricellular rye-grass pollen was observed using the antibodies in gel electrophoresis or enzyme-linked immunosorbent assays.

Unification of the copper(I) binding affinities of the metallo-chaperones Atx1, Atox1, and related proteins : Detection probes and affinity standards

Literature estimates of metal-protein affinities are widely scattered for many systems, as highlighted by the class of metallo-chaperone proteins, which includes human Atox1. The discrepancies may be attributed to unreliable detection probes and/or inconsistent affinity standards. In this study, application of the four CuI ligand probes bicinchoninate, bathocuproine disulfonate, dithiothreitol (Dtt), and glutathione (GSH) is reviewed, and their CuI affinities are re-estimated and unified. Excess bicinchoninate or bathocuproine disulfonate reacts with CuI to yield distinct 1:2 chromatophoric complexes [CuIL2] 3- with formation constants β2 = 1017.2 and 1019.8 M-2, respectively. These constants do not depend on proton concentration for pH ≥7.0. Consequently, they are a pair of complementary and stable probes capable of detecting free Cu+ concentrations from 10-12 to 10-19 M. Dtt binds CuI with KD∼10-15 M at pH 7, but it is air-sensitive, and its CuI affinity varies with pH. The CuI binding properties of Atox1 and related proteins (including the fifth and sixth domains at the N terminus of the Wilson protein ATP7B) were assessed with these probes. The results demonstrate the following: (i) their use permits the stoichiometry of high affinity CuI binding and the individual quantitative affinities (KD values) to be determined reliably via noncompetitive and competitive reactions, respectively; (ii) the scattered literature values are unified by using reliable probes on a unified scale; and (iii) Atox1-type proteins bind CuI with sub-femtomolar affinities, consistent with tight control of labile Cu+ concentrations in living cells.

Detection and defense of application-layer DDoS attacks in backbone web traffic

Web servers are usually located in a well-organized data center where these servers connect with the outside Internet directly through backbones. Meanwhile, the application-layer distributed denials of service (AL-DDoS) attacks are critical threats to the Internet, particularly to those business web servers. Currently, there are some methods designed to handle the AL-DDoS attacks, but most of them cannot be used in heavy backbones. In this paper, we propose a new method to detect AL-DDoS attacks. Our work distinguishes itself from previous methods by considering AL-DDoS attack detection in heavy backbone traffic. Besides, the detection of AL-DDoS attacks is easily misled by flash crowd traffic. In order to overcome this problem, our proposed method constructs a Real-time Frequency Vector (RFV) and real-timely characterizes the traffic as a set of models. By examining the entropy of AL-DDoS attacks and flash crowds, these models can be used to recognize the real AL-DDoS attacks. We integrate the above detection principles into a modularized defense architecture, which consists of a head-end sensor, a detection module and a traffic filter. With a swift AL-DDoS detection speed, the filter is capable of letting the legitimate requests through but the attack traffic is stopped. In the experiment, we adopt certain episodes of real traffic from Sina and Taobao to evaluate our AL-DDoS detection method and architecture. Compared with previous methods, the results show that our approach is very effective in defending AL-DDoS attacks at backbones. © 2013 Elsevier B.V. All rights reserved.

Tunnelling current recognition through core-satellite gold nanoparticles for ultrasensitive detection of copper ions

We report a new method for ultrasensitive detection of Cu(2+), which is based on changes in the tunnelling recognition current across self-assembled core-satellite gold nanoparticles (GNPs) networks functionalised with amino acids (l-cysteine). The addition of copper ions induces the formation of GNP/l-cysteine/Cu(2+)/l-cysteine/GNP molecular junctions and generates a significant decrease in the resistance through the networks. The networks are ultrasensitive to over ten orders range of copper ion concentrations.

Potential-resolved electrogenerated chemiluminescence for the selective detection of multiple luminophores

Electrogenerated chemiluminescence (ECL) is fundamentally dependent on the applied electrode potential, and measuring ECL intensity over a range of different potentials is commonly used to examine the underlying chemical reaction pathways responsible for the emission of light. Several research groups have now demonstrated that the applied potential can be exploited to selectively elicit ECL from: 1) multiple excited states within a single chemical species; 2) multiple emitters sharing a common co-reactant; or 3) distinct ECL systems. This new generation of multiplexed ECL processes has been facilitated by the extensive development of novel electrochemiluminophores and instrumental approaches such as the near-continuous collection of ECL spectra with CCD detectors during voltammetry or chronoamperometry experiments. New dimensions: In electrogenerated chemiluminescence experiments the applied potential can be exploited to selectively elicit light from: multiple excited states within a single chemical species, multiple emitters sharing a common co-reactant, and distinct electrogenerated chemiluminescence systems. These findings may be used to develop low-cost portable analytical devices.