ESI-MS and MALLS analysis of quaternary structure of molluscan hemocyanins

The understanding of the function of macromolecular complexes is mainly related to a precise knowledge of their structure. Recently, the development of suitable mass spectrometric techniques (electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI)) and multi-angle laser light scattering has enabled mass determination of native complexes and of their subunits. By these techniques, the structure and association/dissociation behavior of huge molecules of molluscan Octopus vulgaris, Sepia officinalis and Rapana venosa have been characterized. Molecular masses of the native and dissociated molecule of cephalopodan Hcs O. vulgaris (3545 and 359.3?kDa, respectively) and S. officinalis (4134 and 443.8?kDa, respectively) revealed that only one type subunit organizes their molecules, while the presence of two isoforms with different masses (422.8 and 400.0?kDa) has been determined for gastropodan R. venosa Hc, aggregated into didecamers. The difference of their structural subunits was also established after limited proteolysis with TPCK-trypsin. Eight functional units (FUs) with masses of?~?50?kDa were isolated from both subunits of RvH and isoform of Sepia officinalis, while seven FUs were purified from OvH. Further characterization of proteins by ESI-mass spectrometry (MS) and MALDI-MS, methods gave insights into post-translational modifications such as glycosylation. Glycosylation of O. vulgaris and S. officinalis Hcs was suggested based on the differences (11.6 and 40.0?kDa, respectively) between the masses measured by ESI-MS and those calculated by their gene sequences.     

Positions of the Glycans in Molluscan Hemocyanin, Determined by Fluorescence Spectroscopy

Molluscan hemocyanins are glycoproteins with different quaternary and carbohydrate structures. It was suggested that the carbohydrate chains of some Hcs are involved in their antiviral and antitumor effect, as well in the organization of the quaternary structure of the molecules. Using a well-known complex for saccharide sensing, positions and access to the carbohydrate chains in the native hemocyanins from Rapana venosa (RvH) and Helix lucorum (HlH) and also their structural subunits (RvH1, RvH2 and β_cHlH) and functional units (FUs) were analysed by fluorescence spectroscopy and circular dichroism. Almost no effect was observed in the fluorescence emission after titration of the complex with native RvH and HlH due to lack of free hydroxyl groups which are buried in the didecameric form of the molecules. Titration with the structural subunits β_cHlH and RvH2, increasing of the emission indicates the presence of free hydroxyl groups compared to the native molecules. Complex titration with the structural subunit βc-HlH of H. lucorum Hcs leads to a 2.5 fold increase in fluorescence intensity. However, the highest emission was measured after titration of the complex with FU βcHlH-g. The result was explained by the structural model of β_cHlH-g showing the putative position of the glycans on the surface of the molecule. The results of the fluorescent measurements are in good correlation with those of the circular dichroism data, applied to analyse the effect of titration on the secondary structure of the native molecules and functional units. The results also support our previously made suggestion that the N-linked oligosaccharide trees are involved in the quaternary organization of molluscan Hcs.     

Carbohydrate moieties of molluscan Rapana venosa hemocyanin

Using Zn2+ ions as new method, several FUs have been isolated from molluscan Hc Rapana venosa without formation of non-functional proteolytic side products. N-terminal sequences of these fragments in comparison with FUS from other gastropodan Hcs show a very high degree of structural identity. Four Fus, purified from enzyme-treated structural subunits RvH1 and RvH2 (RvH1-a, RvH1-f, RvH2-a and RvH2-e) show identical N-terminal sequences compared to fragments isolated after treatment with Zn2+ ions. However, in some cases trypsin cleaves RvH chains at different positions if compared to the Zn2+ treatment. To analyze the oligosaccharide composition of two FUS from the first structural subunit of Rapana Hc, RvH1-a and RvH1-f, several techniques were applied: capillary electrophoresis, MALDI-MS, ESI-MS in combination with glycosidase digestions. On basis of these results and the determined amino acid sequence two N-linkage sites were identified in the FU RvH1-a, but only one in the FU RvH1-f.     

Method for Analysis of Different Oligosacchiride Structures

In this study, an improved, rapid, high yield synthesis of N,N’-4,4’-bis(benzyl-2-boronic acid)-bipyridinium dibromide (o-BBV) is described. The obtained o-BVV is applied in a two-component saccharide sensing system (complex) where it serves as a fluorescence quencher and a saccharide receptor. This system was applied to different natural oligosaccharides isolated from molluscan Rapana venosa (RvH1-a) and arthropodan Carcinus aestuarii (CaeH) hemocyanins (Hcs) and cyclodextrins (CDs). The carbohydrate contents of both Hcs were calculated in our previous work to be 1,6 % and 7 % for CaeH and RvH1-a, respectively. We propose that the difference in fluorescence increase of the native CaeH and RvH1-a when titrating them with the complex is due to the fact that the carbohydrate content of CaeH is lower and the carbohydrate chains are buried in between the structural subunits of the native molecule, while the glycans of the functional unit RvH1-a are exposed on the surface of the molecule leading to a 4-fold fluorescence’s intensity change.     

Haliotis tuberculata hemocyanin (HtH): analysis of oligomeric stability of HtH1 and HtH2, and comparison with keyhole limpet hemocyanin KLH1 and KLH2

The multimeric/higher oligomeric states of the two isoforms of Haliotis tuberculata hemocyanin (HtH1 and HtH2) have been assessed by transmission electron microscopy (TEM) of negatively stained specimens, for comparison with previously published structural data from keyhole limpet hemocyanin (KLH1 and KLH2) [see Harris, J.R., Gebauer, W., Guderian, F.U., Markl, J., 1997a. Keyhole limpet hemocyanin (KLH), I: Reassociation from Immucothel followed by separation of KLH1 and KLH2. Micron, 28, 31–41; Harris, J.R., Gebauer, W., Söhngen, S.M., Nermut, M.V., Markl, J., 1997b. Keyhole limpet hemocyanin (KLH). II: Characteristic reassociation properties of purified KLH1 and KLH2. Micron, 28, 43–56; Harris, J.R., Gebauer, W., Adrian, M., Markl, J., 1998. Keyhole limpet hemocyanin (KLH): Slow in vitro reassociation of KLH1 and KLH2 from Immucothel. Micron, 29, 329–339]. In purified samples of both HtH isoforms, the hollow cylindrical ca 8 MDa didecamer predominates together with a small number of decamers, but tri- and longer multidecamers are detectable only in the HtH2. The stability of the two HtH isoforms under varying ionic conditions have been monitored, thereby enabling conditions for the production of stable decamers to be established. The ability of these decamers to reform multimers in the presence of 10 and 100 mM concentrations of calcium and magnesium ions in Tris–HCl buffer (pH 7.4), and also of individual HtH1 and HtH2 subunits (produced by pH 9.6 dissociation in glycine-NaOH buffer), to reassociate in the presence of calcium and magnesium ions, has been assessed. For the HtH1 decamers, the predominant multimeric product is the didecamer at 10 and 100 mM calcium and magnesium concentrations, whereas for the HtH2 decamers, large numbers of multidecamers are produced, with the reaction proceeding more completely at the higher calcium and magnesium concentration. With the HtH1 subunit, reassociation in the presence of 10 and 100 mM calcium and magnesium ions yielded an almost 100% conversion into didecamers, whereas the HtH2 subunit produced a mixture containing large numbers of short multidecamers and relatively few didecamers, together with a considerable number of smaller diameter helical/tubular polymers. The association properties of the HtH1 and HtH2 decamers, and the subunit reassociation, firmly indicate the integrity and structural competency of the protein under the experimental conditions used. Data on the association of KLH2 decamers is also presented, which together with previously published data on the association KLH1 decamers and the reassociation of KLH1 and KLH2 subunits, enables a detailed comparison of the two hemocyanin isoforms from both molluscan species to be made. Biochemical manipulation of the oligomer states and the subunit reassociation of molluscan hemocyanins can usefully be assessed by the study of negatively stained TEM specimens.     

The new physics of ultrathin elastic conoids

Summary Despite centuries of intense effort amongst mathematicians, and a huge literature in this field, there has previously never been a generally valid mathematical model of ultrathin elastic ?shells? (actually surfaces) of revolution. This survey paper presents the first theoretical framework capable of unifying, into a single coherent body of knowledge, a diversity of shapes associated with elastic bows, car bumper-bars, molluscan shells, even flower-buds and pine-cones. It becomes apparent why conventional analysis enjoys limited success when approximating elastic cones to perturbedcylinders anddiscs. Also the paper provides a theoretical basis for analysing the wrinkling of compressed engineering structures. These successes, the new unification and the simplicity of relevant theory which maynever in principle be capable of working in this context.     

Studies on molluscan shells: Contributions from microscopic and analytical methods

Molluscan shells have always attracted the interest of researchers, from biologists to physicists, from paleontologists to materials scientists. Much information is available at present, on the elaborate architecture of the shell, regarding the various Mollusc classes. The crystallographic characterization of the different shell layers, as well as their physical and chemical properties have been the subject of several investigations. In addition, many researches have addressed the characterization of the biological component of the shell and the role it plays in the hard exoskeleton assembly, that is, the biomineralization process. All these topics have seen great advances in the last two or three decades, expanding our knowledge on the shell properties, in terms of structure, functions and composition. This involved the use of a range of specialized and modern techniques, integrating microscopic methods with biochemistry, molecular biology procedures and spectroscopy. However, the factors governing synthesis of a specific crystalline carbonate phase in any particular layer of the shell and the interplay between organic and inorganic components during the biomineral assembly are still not widely known.This present survey deals with microstructural aspects of molluscan shells, as disclosed through use of scanning electron microscopy and related analytical methods (microanalysis, X-ray diffraction, electron diffraction and infrared spectroscopy). These already published data provide relevant information on shells and also contribute for better understanding the biomineralization process.     

Comparative 11A structure of two molluscan hemocyanins from 3D cryo-electron microscopy

Hemocyanins are giant extracellular proteins that transport oxygen in the hemolymph of many molluscs. Molluscan hemocyanins are cylindrical decamers or didecamers of a 350-400 kDa subunit that contains seven or eight different covalently linked globular functional units (FUs), arranged in a linear manner. Each FU carries a single copper active site and reversibly binds one dioxygen molecule. As a consequence, the decamer can carry up to 70 or 80 O(2) molecules. Although complete sequence information is now available from several molluscan hemocyanins, many details of the quaternary structure are still unclear, including the topology of the 10 subunits within the decamer. Here we show 3D reconstructions from cryo-electron micrographs of the hemocyanin decamer of Nautilus pompilius (Cephalopoda) and Haliotis tuberculata (Gastropoda) at a resolution of 11A (FSC(1/2-bit) criterion). The wall structure of both hemocyanins is very similar and shows, as in previous reconstructions, three tiers with 20 functional units each that encircle the cylinder wall, and the 10 oblique minor and major wall grooves. However, the six types of wall FUs of the polypeptide subunit, termed a-b-c-d-e-f, are now for the first time individually discernable by their specific orientation, shape, and connections. Also, the internal collar complex of the decamers shows superior resolution which, in this case, reveals striking differences between the two hemocyanins. The five arcs (FU-g pairs) of the central collar (in both hemocyanins) and the five slabs (FU-h pairs) of the peripheral collar (only present in Haliotis hemocyanin), as well as their connections to the wall and to each other are now more clearly defined. The arc is attached to the wall through a feature termed the anchor, a previously undescribed structural element of the hemocyanin wall.     

基于计算机实测技术的双自由度振动规律验证

由2个劲度系数相同的轻质弹簧与2个质量相同的弹簧振子在竖直方向耦合组成双自由度振动系统.利用计算机实测技术记录了弹簧振子在竖直方向的运动规律,实验值与理论值吻合很好.     

非零位检测凸非球面反射镜的研究

在简要总结各种检测凸非球面方法优缺点的基础上,提出了利用部分补偿法和子孔径拼接干涉检测凸非球面的新方法。分别研究和分析了这两种非零位检测非球面方法的基本原理和基础理论;设计并制作了部分补偿器件,并对其系统误差进行了标定;开发了综合优化和误差均化的子孔径拼接算法;设计并研制了两种方法都适用的检测装置。并结合实例对一口径为130 mm的碳化硅凸非球面分别进行了部分补偿检测和子孔径拼接测量,这两种方法测量所得的全口径面形分布是一致的,其PV值和RMS值的偏差仅为0.010 和 0.002 (=0.632 8 m)。从而提供了两种非零位补偿测试凸非球面的手段。     

Influence of purity of HfO2 on reflectance of ultraviolet multilayer

The impurities in two kinds of HfO2 materials and in their corresponding single layer thin films were determined through glow discharge mass spectrum technology and secondary ion mass spectrometry(SIMS) equipment respectively.It was found that ZrO2 was the main impurity in the two kinds of HfO2 either in the original HfO2 materials or in the electron beam deposited films.In addition,the difference of Zr content in the two kinds of HfO2 single laver films was much laxger than that of the other impurities such as Ti and Fe.which showed that it was just ZrO2 that made the difference between the optical performance of the film products including the two kinds of HfO2.With these two kinds of HfO2 and the same kind of SiO2.we deposited HfO2/SiO2 multilayer reflective coatings at the wavelength of 266 nm.Experimental results showed that the reflectances of these two mirrors were about 99.85% and 99.15% respectively,which agreed well with the designed results what were based on the optical constants obtained from the corresponding single layer thin films.     

微生物传感器BOD测定方法的适应性及分析要点

详细论述了微生物传感器BOD测定方法的适应性和分析要点,并在具体的实践过程中摸索出两种更为有效的活化微生物膜的方法,此两种方法与传统的膜活化方法相比,具有活化时间短、仪器状态和测量数据稳定等优点,更为适合在实际测定BOD中的应用。     

无衍射贝塞尔光束相干的理论与实验

 分别使用两种不同底角的轴棱锥产生无衍射贝塞尔光束。理论分析了单束贝塞尔光及两束贝塞尔光相干叠加后光场的光强分布,分析了产生的局域空心光束在一个完整周期内的特性。数值模拟了光沿纵向传播时光场的横向光强分布,并给出了相关的实验。对单束贝塞尔光及两束贝塞尔光干涉后的中心光斑的大小进行了测量,测量结果与理论计算结果吻合。     

不同电介质结构下介质阻挡放电特性研究

设计制作了单面有氧化铟锡(ITO)导电介质层的双玻璃介质层的介质阻挡放电装置,研究了其放电特性,并将其与双玻璃介质层和单玻璃介质层的介质阻挡放电进行了比较.从电荷输运的角度分析,上述三种装置分别实现了电荷的二维、零维和三维输运.采用两种不同的双玻璃介质层装置,获得了单个稳定的放电丝.与无ITO导电层的双玻璃结构得到的单个放电丝相比,单面有导电ITO介质的双玻璃结构中,单放电丝呈"T"字型,其光晕是前者光晕的2倍,其放电电流大于前者电流,其放电时间间隔长短交替现象更为明显,且存在强度大小交替的现象.分析表明,壁电荷输运及二次电子发射的不同导致了不同电介质结构放电特性的不同. 关键词： 介质阻挡放电 壁电荷 二次电子发射     

Investigations of three-dimensional flow characteristics in a liquid ramjet combustor using the PIV method

Three-dimensional flow characteristics in a liquid fuel ramjet combustor were investigated using the PIV method. The combustor had two rectangular inlets that form a 90-degree angle with each other, with intake angles of 30 degrees. Three guide vanes were installed in each rectangular inlet to improve flow stability. The experiments were performed in a water tunnel test with the same Reynolds number as Mach 0.3 at the inlet. PIV software was developed to measure the characteristics of the flow field in the combustor. Accuracy of the developed PIV program was verified with a rotating disk experiment and standard data. The experimental results showed that the two main streams from the rectangular intakes collided near the plane of symmetry and generated two large longitudinal vortices, which was in agreement with three dimensional computational results. A large and complex threedimensional recirculating flow was measured behind the intakes.     

Lateralization and detection of pulse trains with alternating interaural time delays

The effect of onset interaural time differences (ITDs) on lateralization and detection was investigated for broadband pulse trains 250 ms long with a binaural fundamental frequency of 250 Hz. Within each train, ITDs of successive binaural pulse pairs alternated between two of three values (0 micros, 500 micros left-leading, and 500 micros right-leading) or were invariant. For the alternating conditions, the experimental manipulation was the choice of which of two ITDs was presented first (i.e., at stimulus onset). Lateralization, which was estimated using a broadband noise pointer with a listener adjustable interaural delay, was determined largely by the onset ITD. However, detection thresholds for the signals in left-leading or diotic continuous broadband noise were not affected by where the signals were lateralized. A quantitative analysis suggested that binaural masked thresholds for the pulse trains were well accounted for by the level and phase of harmonic components at 500 and 750 Hz. Detection thresholds obtained for brief stimuli (two binaural pulse or noise burst pairs) were also independent of which of two ITDs was presented first. The control of lateralization by onset cues appears to be based on mechanisms not essential for binaural detection.     

Indirect Determination of Electronic Transition Frequencies of Coronene,Triphenylene and 1,2,5,6-Dibenzanthracene in the Vapor Phase

Frequencies of absorption band maxima for the first two electronic singlet transitions of coronene, triphenylene and 1,2,5,6-dibenzanthracene in vapor phase were calculated indirectly from solution spectral data. The calculations were based on two different models: the first one was a linear correlation between the solution absorption frequencies in nonpolar solvents and the solvent refractive indices. The accuracy of both methods was checked with anthracene and some of its derivatives: both methods gave a good agreement with the experimental values.     

两种不同前处理方法测定山茶油中的痕量铜

采用湿法消解和萃取法两种不同的前处理方法,石墨炉原子吸收光谱法测定山茶油中精炼油和毛油中痕量的铜,结果表明:两种方法有较好的一致性,湿法消解在精密度上要优于萃取法,萃取法较湿法消解耗时短,试剂消耗少。两种方法的回收率在82.5%—114.5%之间,湿法消解的检出限为0.0134μg/g,萃取法的检出限为0.0160μg/g。     

Effects of collision between two plumes on plume expansion dynamics during pulsed laser ablation in background gas

Si and Ge targets were simultaneously irradiated by individual two pulsed lasers, and two plumes from the targets were collided head-on with expectation to prepare hybrid nanoparticles. We investigate effects of He background gas pressure on plume collision dynamics. Three characteristic behaviors of plume expansion dynamics are observed at low, middle, and high background gas pressure regions. Interaction between the two atomic species during plume expansion was small and the effect of collision was hardly observed at a low background gas pressure, 130 Pa, while spatial evolution of the plume was suppressed at middle pressure, 270 Pa, due to collision of the two plumes. At high pressure, 2700 Pa, plume expansion is suppressed by background gas and the effect of a direct collision of two plumes was small. These results indicate that plume collision dynamics, which governs nanoparticle formation, and the mixture of Si and Ge species can be varied by background gas pressure. The deposit near the center of two targets was nanoparticles that were composed of Si and Ge.     

Density functional study of zigzag BN nanotubes with equivalent ends

A density functional theory (DFT) study was performed on representative model of zigzag boron nitride nanotubes (BNNTs) with equivalent ends. Two models of (6,0) BNNTs were considered in the calculations in which a belt composed of carbon atoms was substituted instead of boron and nitrogen atoms in the middle of the nanotube. Hence, model 1 was created with two equivalent B-ends and model 2 was created with two equivalent N-ends. The optimization process and also the calculated electric field gradient (EFG) tensors in two models of BNNT remarkably revealed that the electronic structure properties of those nuclei located at the end of nanotube are duplicated in the considered models of BNNTs. The calculations were performed at the level BLYP method and 6-31G* standard basis set using GAUSSIAN 98 package of program.