Discovery of the ammonium substrate site on glutamine synthetase, a third cation binding site

Glutamine synthetase (GS) catalyzes the ATP-dependent condensation of ammonia and glutamate to yield glutamine, ADP, and inorganic phosphate in the presence of divalent cations. Bacterial GS is an enzyme of 12 identical subunits, arranged in two rings of 6, with the active site between each pair of subunits in a ring. In earlier work, we have reported the locations within the funnel-shaped active site of the substrates glutamate and ATP and of the two divalent cations, but the site for ammonia (or ammonium) has remained elusive. Here we report the discovery by X-ray crystallography of a binding site on GS for monovalent cations, Tl+ and Cs+, which is probably the binding site for the substrate ammonium ion. Fourier difference maps show the following. (1) Tl+ and Cs+ bind at essentially the same site, with ligands being Glu 212, Tyr 179, Asp 50', Ser 53' of the adjacent subunit, and the substrate glutamate. From its position adjacent to the substrate glutamate and the cofactor ADP, we propose that this monovalent cation site is the substrate ammonium ion binding site. This proposal is supported by enzyme kinetics. Our kinetic measurements show that Tl+, Cs+, and NH4+ are competitive inhibitors to NH2OH in the gamma-glutamyl transfer reaction. (2) GS is a trimetallic enzyme containing two divalent cation sites (n1, n2) and one monovalent cation site per subunit. These three closely spaced ions are all at the active site: the distance between n1 and n2 is 6 A, between n1 and Tl+ is 4 A, and between n2 and Tl+ is 7 A. Glu 212 and the substrate glutamate are bridging ligands for the n1 ion and Tl+. (3) The presence of a monovalent cation in this site may enhance the structural stability of GS, because of its effect of balancing the negative charges of the substrate glutamate and its ligands and because of strengthening the "side-to-side" intersubunit interaction through the cation-protein bonding. (4) The presence of the cofactor ADP increases the Tl+ binding to GS because ADP binding induces movement of Asp 50' toward this monovalent cation site, essentially forming the site. This observation supports a two-step mechanism with ordered substrate binding: ATP first binds to GS, then Glu binds and attacks ATP to form gamma-glutamyl phosphate and ADP, which complete the ammonium binding site. The third substrate, an ammonium ion, then binds to GS, and then loses a proton to form the more active species ammonia, which attacks the gamma-glutamyl phosphate to yield Gln. (5) Because the products (Glu or Gln) of the reactions catalyzed by GS are determined by the molecule (water or ammonium) attacking the intermediate gamma-glutamyl phosphate, this negatively charged ammonium binding pocket has been designed naturally for high affinity of ammonium to GS, permitting glutamine synthesis to proceed in aqueous solution.     

A systematic mapping of semi-formal and formal methods in requirements engineering of industrial Cyber-Physical systems

Journal of Intelligent Manufacturing - The requirements engineering of Industrial Cyber-Physical Systems is extremely challenging due to large system sizes, component heterogeneity, involvement of...     

Noninvasive glucose monitoring in vivo with an optical heterodyne polarimeter

An amplitude-sensitive optical heterodyne polarimeter was set up to monitor noninvasively the aqueous glucose concentration in a rabbit's eye. A Zeeman laser in conjunction with a Glan-Thompson analyzer was used to generate an optical heterodyne signal. The amplitude of the heterodyne signal linearly related to the optical rotation angle of the aqueous glucose. The concentration of the aqueous glucose in a rabbit's eyeball was measured in vivo. There was a 30-min time delay between observations of aqueous glucose and blood glucose. The detection capability and the reproducibility of the experiment are demonstrated and discussed.     

Random-iteration algorithm-based optical parallel architecture for fractal-image decoding by use of iterated-function system codes

An optical parallel architecture for the random-iteration algorithm to decode a fractal image by use of iterated-function system (IFS) codes is proposed. The code value is first converted into transmittance in film or a spatial light modulator in the optical part of the system. With an optical-to-electrical converter, electrical-to-optical converter, and some electronic circuits for addition and delay, we can perform the contractive affine transformation (CAT) denoted in IFS codes. In the proposed decoding architecture all CAT's generate points (image pixels) in parallel, and these points then are joined for display purposes. Therefore the decoding speed is improved greatly compared with existing serial-decoding architectures. In addition, an error and stability analysis that considers nonperfect elements is presented for the proposed optical system. Finally, simulation results are given to validate the proposed architecture.     

Pressure infiltration of sol/gel processed short fibre ceramic matrix composites

Using pressure infiltration short fibre-reinforced ceramic matrix composites with uniform and dense microstructure can be made. Based upon this processing technique, composites composed of silica sol, alumina particles, and alumina short fibres were fabricated. The related processing parameters studied in this work include infiltration rate, fibre volume fraction Vf, particle size, and infiltrate viscosity. An optimal infiltration rate was 4 mm min-1, at which rate the composite with Vf of 8.1% and particle size of 3 m has the highest green density. An equilibrium between particle packing strength and applied load must be obtained during the infiltration to obtain high green density and composite strength. The influence of fibre volume fraction and particle size on composite green density is in a synergistic manner because it involves particle–fibre interactions, fibre–fibre interactions, and sedimentation. Furthermore, the increase of sol viscosity results in more sedimentation in the infiltrate and lower composite green density. The fracture toughness of composites is 38% higher than that of monolithic alumina. © 1998 Chapman & Hall     

Ho:CaF(2) solid-state saturable-absorber Q switch for the 2-µm Tm,Cr:Y(3)Al(5)O(12) laser

The holmium-doped calcium fluoride (Ho:CaF(2)) crystal was shown to be an effective solid-state saturable-absorber Q-switch for a flash-lamp-pumped Tm,Cr:Y(3)Al(5)O(12) laser at 2.017 μm. With a 1-cm-thick Ho(0.5%),Er(5%):CaP(2) saturable absorber and a 6.3% output coupler, a single Q-switched laser pulse of 51 mJ in energy and 60 ns in duration was obtained at a flash-lamp input energy of 85 J. With a 14.6% output coupler, a typical Q-switched laser pulse of 84 mJ and 82 ns was observed.     

高速数据采集系统的设计与实现

由于雷达数据处理的要求,需要对雷达数据进行实时地存储记录。本文介绍了一种基于光纤通道的高速数据采集系统,以FPGA为平台,PCIExpress为接口,链式DMA为传输模式,实现了最高速率为6Gbps的数据采集系统。     

A template-based baseball video scene classification using efficient playfield segmentation

In this paper, we present an effective and efficient framework for baseball video scene classification. The results of scene classification can be able to provide the ground for baseball video abstraction and high-level event extraction. In general, most conventional approaches are shot-based, which shot change detection and key-frame extraction are necessary prerequisite procedures. On the contrary, we propose a frame-based approach. In our scene classification framework, an efficient playfield segmentation technique is proposed, and then the reduced field maps are utilized as scene templates. Because the shot change detection and the key-frame extraction are not required in proposed method, the new framework is very simple and efficient. The experimental results have demonstrated that the effectiveness of our proposed framework for baseball videos scene classification, and it can be easily extended the template-based approach to other kinds of sports videos.     

Simulation optimization using particle swarm optimization algorithm with application to assembly line design

Assembly line design is an important part of production system. Some processes need to undergo changes in order to increase in efficiency. Computer simulation has been applied on process design for many decades. Traditionally, simulation had to run all possible alternatives of assembly line and was not considered as an optimization technique. Thus, this study employs particle swarm optimization (PSO) algorithm which is with mutation based on similarity for simulation optimization in order to optimize the managerial parameters in production system. Through experimentation designs and statistics tests, the simulation results show that the proposed method is better than other algorithms, like genetic algorithm and conventional PSO algorithm for solving assembly line design problem.