蔗糖梯度离心法纯化Vero细胞乙脑疫苗的纯度分析及与层析法的比较

通过对蔗糖梯度离心法纯化Vero细胞乙脑疫苗的纯化工艺进行分析,发现现行工艺中收取的病毒组分中,不同蔗糖浓度中病毒的纯度是不同的,而呈峰型且与病毒效力峰及蛋白浓度峰不重合。实验结果对今后工艺的改进具有指导意义。另外应用Sephacryl-s-1000凝胶层析法对乙脑病毒进行了纯化研究和分析,发现蔗糖梯度离心法和层析法纯化的病毒的纯度及回收率分别为291．46u／mg、96．01％和309．41 u／mg 65．08％,Sephacryl-s-1000凝胶层析法同样可以获得较高的纯度和收率。     

Synthesis and complexation properties towards the ammonium cation of aza-coronand analogues containing sucrose

1',2,3,3',4,4'-Hexa-O-benzyl-sucrose was converted in good yields into the macrocyclic receptors containing two and three nitrogen atoms in the ring. Their complexation properties towards the ammonium cation were significantly higher than for receptors without any nitrogen atoms in the ring.     

Pressure dependence on electronic structures,charge distribution and bond orders of solid nitromethane using nonlocal DFT functional

The electronic properties of solid nitromethane are studied using nonlocal exchange-correlation functional (optPBE–vdW) under hydrostatic compression up to 40?GPa. We found that the optPBE–vdW functional can reproduce well the crystalline structures compared with the experiments, and an isomorphic phase transition has been verified by their P–V curve. Bader’s charge analysis shows the electron flows from CH₃ group to NO₂ group with the pressure. Moreover, the calculated bond orders show that the pressure only strengthens the intermolecular C–N bond and intermolecular C–H···O hydrogen bonds though it shortens all bond lengths. Furthermore, the electronic structure and its pressure dependence have also been discussed in detail.     

Octanol-water partition coefficient of glucose, sucrose, and trehalose

The octanol-water partition coefficients (P) of glucose, sucrose, and trehalose were measured at temperatures between 5 and 20 degrees C using an enzymatic method. The measured log P is compared with calculated and experimental data previously reported. In the case of trehalose, the measured log P differs considerably from the theoretically estimated values and agrees with the expected value for a disaccharide. Some methods of assessing the partition coefficients are also analyzed and it is concluded that the atom/fragment contribution method overestimates the hydrophilicity of disaccharides and, probably in a larger extension, that of trisaccharides. The knowledge of P for these sugars is valuable both for basic or applied purposes, including food and biomolecules stabilization.     

Effect of blood storage on erythrocyte/wall interactions: implications for surface charge and rigidity

In this report, we study, under flow conditions, the interactions of stored erythrocytes with an artificial surface: a microelectrode whose charge density ranges from –15 to +27 μC/cm². Interactions consist of red cells slowly circulating on the microelectrode and exerting a real contact with the electrode. Interaction is detected and measured by transient fluctuations of the electrolyte resistance obtained by impedance measurement of the microelectrode. Effects of aging induced by storage of whole blood at 4 °C show that the surface charge of erythrocytes rapidly decreases when blood is stored for more than 6 days under our experimental conditions. In comparison with trypsin-treated erythrocytes, an eight day storage induces a 60% decrease in the surface charge of red cells. After two weeks of storage, red cells are no longer negatively charged, presumably be cause of removal of sialic acid. Cells rigidity is significant after 6 days of storage and influences the electrical contact. Membrane rigidity increase could arise from the surface charge decrease. Finally, the surface charge decrease could be of importance in the use of stored blood. Received: 30 October 1996 / Accepted: 12 November 1996     

Understanding atomic bonding and electronic distributions of a DNA molecule using DFT calculation and BOLS-BC model

Deoxyribonucleic acid (DNA) is an important molecule that has been extensively researched, mainly due to its structure and function. Herein, we investigated the electronic behavior of the DNA molecule containing 1008 atoms using density functional theory. The bond-charge (BC) model shows the relationship between charge density and atomic strain. Besides, the model mentioned above is combined with the bond-order-length-strength (BOLS) notion to calculate the atomic cohesive energy, the bond energy, and the local bond strain of the DNA chain. Using the BOLS-BC model, we were able to obtain information on the bonding features of the DNA chain and better comprehend the associated properties of electrons in biological systems. Consequently, this report functions as a theoretical reference for the precise regulation of the electrons and the bonding states of biological systems.     

Regioselective sulfonylation of 6,1',6'-tri-O-tritylsucrose through dibutylstannylation: synthesis of 4'-O-sulfonyl derivatives of sucrose

3-O-Mesyl-1,6-di-O-trityl-beta-D-fructofuranosyl-(2-->1)-6-O-trityl-alpha-D-glucopyranoside (3) was synthesized via stannylation of 6,1',6'-tri-O-tritylsucrose with dibutyltin oxide in benzene, followed by treatment of the crude product with methanesulfonyl chloride in the presence of triethylamine in dichloromethane at 0 degrees C. A similar treatment of the tri-tritylsucrose in toluene, instead of benzene, yielded 4-O-mesyl-1,6-di-O-trityl-beta-D-fructofuranosyl-(2-->1)-6-O-trityl-alpha-D-glucopyranoside (4) as the major product. The X-ray crystal structure of the corresponding acetyl derivative, 3-O-acetyl-4-O-mesyl-1,6-di-O-trityl-beta-D-fructofuranosyl-(2-->1)-2,3,4-tri-O-acetyl-6-O-trityl-alpha-D-glucopyranoside (5), confirms the position and stereochemistry of the methanesulfonyl group at C-4 of the fructofuranosyl ring.     

Interactions of erythrocytes with an artificial wall: influence of the electrical surface charge

Electrical charge on any biological surface plays a crucial role in its interaction with other molecules or surfaces. Here, we study, under flow conditions, the interactions of erythrocytes with an artificial surface: a platinum microelectrode whose charge density ranges from –15 to +27 μC/cm². This artificial surface could be similar in surface charge to an endothelium or a biomaterial. In this model, interactions are measured as a transient relative increase of the electrolyte resistance obtained by impedance measurement of a microelectrode. A maximal interaction of erythrocytes with the charged surface is calculated in the 0 to +10 μC/cm² charge density range. At negative surface charge, a less efficient contact was obtained because of electrostatic repulsion forces. High positive surface charge (charge density >10 μC/cm²) does not improve the contact but induces a progressive decrease in the contact efficiency, which could be explained by a rearrangement of macromolecules on the erythrocyte surface or an effect of positive groups on the cell membrane. This work suggests that a greater surface area of contact is obtained in the 0 to +10 μC/cm² charge density range and that this is provided by more molecular bridges. Received: 23 February 1996 / Accepted: 26 April 1996     

Mapping of putative quantitative trait loci controlling the total oligosaccharide and sucrose content of Glycine max seeds

Although oligosaccharides and sucrose are very important nutritional components of soybean seeds, little information is available about inheritance of oligosaccharide and sucrose content. The objective of this study was to identify quantitative trait loci (QTLs) that determine the oligosaccharide and sucrose content of soybean. The 117 F_2:10 recombinant inbred lines developed from a cross of “Keunolkong” and “Shinpaldalkong” were used. Narrow-sense heritability estimates, on a plot mean basis, of oligosaccharide and sucrose content were 79.07 and 74.84%, respectively. Four QTLs for oligosaccharide content were located on linkage groups (LG) C2, H, J, and L. Sucrose content was related with two QTLs located on LG H and J. Total oligosaccharide and sucrose content have two common QTLs on LG H and J.     

Probing the effect of nitro groups in nitramine based energetic molecules: a DFT and charge density study

The structure, electron density distribution, energetic and electrostatic properties of simple nitramine based energetic TMA, DMNA, MDA and TNA molecules were determined using density functional theory (B3LYP) with the 6-311G^** and aug-cc-pVDZ basis sets coupled with Bader's theory of atoms in molecules. In the NO₂ group substituted molecules, the N–N bond distance increases with the increase of NO₂ groups, whereas in C–N bonds, this effect is relatively less, and the distances are almost equal. The topological analysis of electron density reveals that the electron density ρ_bcp(r) of C–N and N–N bonds are significantly decreasing with the increase of NO₂ groups in the nitramine molecules. The Laplacian of electron density ▽²ρ_bcp(r) of N–NO₂ bonds [DMNA: ? 16.7 eÅ^? 5, MDA: ? 12.8 eÅ^? 5 and TNA: ? 7.9 eÅ^? 5] of the molecules are relatively less negative, and the values also decrease with the increase of NO₂ groups; this implies that the charge concentration decreases with the increase of NO₂ groups, which leads to weakening the N–N bonds of the molecules. The isosurface of molecular electrostatic potential displays high electronegative regions around the NO₂ groups. The oxygen balance OB₁₀₀ of the molecules increases as the number of NO₂ group increases in the molecules, in which, the TNA molecule having maximum OB₁₀₀ value [+7.89]. The band gap, heat of detonation, bond dissociation energy and charge imbalance are predominantly depends on the number of NO₂ group present in the molecule. The charge imbalance parameter (ν) has been calculated for all molecules, which reveals that TNA is a highly sensitive molecule, the corresponding ν value is 0.047.     

Optical properties of silver(I) perrhenate: luminescence from a metal-to-metal charge transfer state

The salt Ag⁺ReO₄ ⁻ shows an orange luminescence (λ_max=580 nm), which originates from a Ag^I → Re^VII MMCT triplet.     

Investigation of intermolecular interactions and stability of verubecestat in the active site of BACE1: Development of first model from QM/MM-based charge density and MD analysis

Alzheimer disease (AD) is a cruel neurodegenerative disorder caused by the deposition of amyloid β (Aβ) peptide inside the brain. The β-secretase (beta amyloid precursor protein (APP) cleaving enzyme 1, BACE1) is one of the enzymes involved in the cleavage of APP that leads to the Aβ formation and it is the primary target for the treatment of AD. Recent report outlines that verubecestat molecule strongly inhibits BACE1; however, its structure, binding mechanism and the stability in the active site of BACE1 are not yet known. The present study aims to determine the structure, binding affinity and the stability of verubecestat molecule in the active site of BACE1 from the molecular docking, quantum mechanics/molecular mechanics (QM/MM)-based charge density analysis and molecular dynamics simulation. Verubecestat molecule was docked at BACE1; it shows high binding affinity towards BACE1. Further, the conformational geometry and the intermolecular interactions of verubecestat in the active site of BACE1 were determined. The molecule forms strong interaction with the neighboring amino acids in the active site of BACE1. The onsite QM/MM-based charge density analysis reveals the nature of charge density distribution and the topological properties of intermolecular interactions of verubecestat molecule in the active site of BACE1. The calculated electrostatic potential (ESP) of verubecestat in the active site of BACE1 displays high negative and positive ESP regions of the molecule. This onsite QM/MM analysis is more relevant to the physiological situation. The molecular dynamics simulation has been performed, which confirms the high stability and compactness of verubecestat in the active site of BACE1. The MM-generalized Born surface area and MM-Poisson Boltzmann surface area free energy calculations of verubecestat–BACE1 also confirm the high binding affinity of verubecestat.

Communicated by Ramaswamy H. Sarma     

Surface charge density estimation by 9-aminoacridine fluorescence titration: improvements and limitations

A large number of surface charge density () and surface potential (_o) estimations have been based on 1) titrations of the fluorescence of 9-aminoacridine released from the diffuse double layer adjacent to negatively charged membrane surfaces by non-adsorbing monovalent and divalent cations, and 2) calculations using experimental data from the titration curves and the Gouy-Chapman theory of the diffuse double layer. In this paper we discuss the different simplifying approximations employed in the earlier calculations and recommend modified formulas for the calculations. The latter have been derived without any simplifying approximation concerning the ionic (electrolyte) composition of the titration assays. We also show that depends, to some extent, on the concentrations of buffer and vesicles in the assays and present experimental evidence that decamethonium (decane-1,10-bis-trimethylammonium), a bulky organic divalent cation, can be satisfactorily used for the estimation of under well-defined conditions, despite its putative interaction with membranes.Abbreviations 9-AA 9-aminoacridine - (DeM)²⁺ decamethonium - (DiM)²⁺ dimethonium - EDTA ethylenediaminetetraacetic acid - EGTA ethylene glyol-bis(-aminoethyl ether) N,N,N,N-tetraacetic acid - (HeM)²⁺ hexamethonium - MES 2-(N-morpholino) ethanesulfonic acid - MOPS 4-morpholinopropanesulfonic acid - PM plasma membrane - Tris tris(hydroxymethyl)aminomethane - surface charge density - _o surface potential Correspondence to: A. Bérczi     

Understanding the conformational flexibility and electrostatic properties of curcumin in the active site of rhAChE via molecular docking,molecular dynamics,and charge density analysis

Acetylcholinesterase (AChE) is an important enzyme responsible for Alzheimer’s disease, as per report, keto-enol form of curcumin inhibits this enzyme. The present study aims to understand the binding mechanism of keto-enol curcumin with the recombinant human Acetylcholinesterase (rhAChE) from its conformational flexibility, intermolecular interactions, charge density distribution, and the electrostatic properties at the active site of rhAChE. To accomplish this, a molecular docking analysis of curcumin with the rhAChE was performed, which gives the structure and conformation of curcumin in the active site of rhAChE. Further, the charge density distribution and the electrostatic properties of curcumin molecule (lifted from the active site of rhAChE) were determined from the high level density functional theory (DFT) calculations coupled with the charge density analysis. On the other hand, the curcumin molecule was optimized (gas phase) using DFT method and further, the structure and charge density analysis were also carried out. On comparing the conformation, charge density distribution and the electrostatic potential of the active site form of curcumin with the corresponding gas phase form reveals that the above said properties are significantly altered when curcumin is present in the active site of rhAChE. The conformational stability and the interaction of curcumin in the active site are also studied using molecular dynamics simulation, which shows a large variation in the conformational geometry of curcumin as well as the intermolecular interactions.     

Heteronuclear coupling constants of hydroxyl protons in a water solution of oligosaccharides: trehalose and sucrose

Relatively few details are known about the conformational preferences of hydroxyl groups in carbohydrates in water solution, though these would be informative about solvation and H-bonding. We show that highly concentrated solutions of sucrose and trehalose exhibit surprisingly well-resolved ¹H NMR spectra in a deuterium oxide–water solvent mixture at subzero temperatures. Measurement conditions are suitable to extract nearly all homonuclear and, for the first time, heteronuclear coupling constants of OH groups of carbohydrates in their natural abundance. For ^2,3J_HO,C coupling constants new, powerful variants of HETLOC and HECADE techniques were applied. The present data do not support the presence of persistent H-bonds in these two cryogenic disaccharides.     

Effects of cold immobilization and recovery period on honeybee learning, memory, and responsiveness to sucrose

In addition to human error and variation in laboratory conditions, there are numerous factors that can complicate comparisons among studies. Furthermore, differences in how experimental methods are executed can make it difficult to distinguish between effects of focal versus extraneous variables. Insect neural function is commonly evaluated using Pavlovian conditioning techniques; learning and memory in many species can be assessed using the proboscis extension reflex (PER). However, there are significant inconsistencies in methods used to immobilize insects prior to PER tests. We compared responses of honeybees immobilized in a refrigerator, on ice, and in a freezer, and evaluated influence of recovery interval before testing. Ice-chilling weakly decreased learning (response to an originally neutral odor) more so than refrigeration or freezing, but not 24-h recall of odor. We found no significant differences in responsiveness to sucrose relative to cooling method, but responsiveness was significantly lower among honeybees left to recover for only 0.75 h versus 1.5 or 3 h. Finally, we observed increased responsiveness to sucrose and geraniol between June and August. Our results suggest that inconsistencies in cold immobilization methods could confound interpretation and comparison of results from a large body of work on honeybee learning and memory.     

De-novo synthesis of fructans from sucrose in vitro by a combination of two purified enzymes (sucrose: sucrose 1-fructosyl transferase and fructan: fructan 1-fructosyl transferase) from chicory roots (Cichorium intybus L.)

Although fructans occur widely in several plant families and they have been a subject of investigation for decennia, the mechanism of their biosynthesis is not completely elucidated. We succeeded in purifying a fructan: fructan 1-fructosyl transferase (1-FFT; EC 2.4.1.100) from chicory roots (Cichorium intybus L. var. foliosum cv. Flash). In combination with the purified chicory root sucrose: sucrose 1-fructosyl transferase (1-SST; EC 2.4.1.99), this enzyme synthesized a range of naturally occurring chicory fructans (inulins) from sucrose as the sole substrate. Starting from physiologically relevant sucrose concentrations, inulins up to a degree of polymerization (DP) of about 20 were synthesized in vitro after 96 h at 0°C. Neither 1-SST, nor 1-FFT alone could mediate the observed fructan synthesis. Fructan synthesis in vitro was compared starting from 50, 100 and 200 mM sucrose, respectively. The initiation of (DP > 3)-fructan synthesis was found to be correlated with a certain ratio of 1 kestose to sucrose. The data presented now provide strong evidence to validate the 1-SST/1-FFT model for in-vivo fructan synthesis, at least in the Asteraceae.Abbreviations DP degree of polymerization - 1-FFT fructan: fructan 1-fructosyl transferase - 1-SST sucrose: sucrose 1-fructosyl transferase The authors thank E. Nackaerts for valuable technical assistance. W. Van den Ende is grateful to the National Fund for Scientific Research (NFSR Belgium) for giving a grant for research assistants.     

Effects of inoculum density,zeatin and sucrose on anthocyanin accumulation in a carrot suspension culture

Anthocyanin formation in a suspension culture of Daucus carota is induced by transfer from medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) to one lacking 2,4-D. The specific yields were strongly influenced by the inoculum density. Inoculum density altered the effect of zeatin concentration on anthocyanin accumulation. The in part by increasing the sucrose levels. It was inferred from the results that sucrose was exhausted at a low concentration of sucrose and at a high cell density, resulting in the decrease of yield of anthocyanin.