0．23T稳恒磁场对不同温度离体过氧化氢酶的磁效应研究

研究了 0 .2 3T稳恒磁场对不同温度下的离体牛肝过氧化氢酶 (CAT)构象及活力的影响 ,并从分子水平讨论了磁场对不同温度的过氧化氢酶产生不同生物学效应的可能机制。将不同温度的天然酶液置于磁感应强度为0 .2 3T的磁场中分别处理一定的时间 ,处理过程中保持环境温度与酶液温度一致 ,撤离磁场后立即在相同实验条件下对其进行光谱分析及量热分析 ,并用Beers&Sizers法 (改良型 )测定酶活力。结果表明 ,磁场使 2 5℃过氧化氢酶的构象发生明显变化 ,表现为荧光偏振度增加、出现明显的差示扫描量热曲线、产生λ2 10nm～ 310nm的紫外差光谱以及λ330nm荧光发射峰的荧光强度改变 (荧光发射峰的峰位未移动 ) ,构象变化的同时酶活力增加 ;15℃过氧化氢酶的构象及活力变化规律与 2 5℃过氧化氢酶类似 ,但强度均弱于 2 5℃酶 ;而 4℃过氧化氢酶的构象及活力没有发生变化 ,表现出未受磁场处理的影响。相同实验条件下 ,磁场对不同温度的酶分子影响不同 ,随温度的增加 ,影响效应趋于显著。由于不同温度的酶分子之间的差异在于构象状态的不同 ,这表明酶分子自身的构象状态对磁场处理效果有极其重要的影响。不同温度的过氧化氢酶磁效应差异显著可能是由磁致酶构象变化的特殊机制所引起。磁场对酶分子构象的影响可能是通     

核磁共振技术在蛋白质领域的应用

简要叙述了核磁共振技术(NMR)在蛋白质领域的研究及应用。NMR法通过测定蛋白质在稀溶液状态下反应位点的特定参数来计算蛋白质的三级结构,并可深入了解一定时间范围内化学反应和蛋白质构象转变的动力学过程。通过NMR对抗原决定簇和抗体CDR作图,可以分析其一级结构和三维构象;对抗原抗体动力学的分析,对于设计基因疫苗、检测细胞表面抗原提呈以及分析抗原抗体复合物的构象变化也有着重要意义。     

Effects of hypomagnetic field on noradrenergic activities in the brainstem of golden hamster

Previous studies found that elimination of the geomagnetic field (GMF) interferes with the normal brain functions, but the underlying mechanism remains unknown. The present study examined the effects of long-term exposures to a near-zero magnetic environment on the noradrenergic activities in the brainstem of golden hamsters. Both the content of norepinephrine (NE) and the density of NE-immunopositive neurons in the tissue decreased significantly after the treatment, and the effects could be progressive with time. These variations may substantially contribute to behavioral and mood disorders reported in other studies when animals are shielded from the GMF.     

Crystal structure of a bacterial albumin-binding domain at 1.4 A resolution

The albumin-binding domain, or GA module, of the peptostreptococcal albumin-binding protein expressed in pathogenic strains of Finegoldia magna is believed to be responsible for the virulence and increased growth rate of these strains. Here we present the 1.4A crystal structure of this domain, and compare it with the crystal structure of the GA-albumin complex. An analysis of protein-protein interactions in the two crystals, and the presence of multimeric GA species in solution, indicate the GA module is "sticky", and is capable of forming contacts with a range of protein surfaces. This might lead to interactions with different host proteins.     

In Vitro Culture of Phytomonas Sp. Isolated from Euphorbia characias. Metabolic Studies by 1H NMR

ABSTRACT. We describe the in vitro culture of Phytomonas species isolated from Euphorbia characias . The best choice between tested media was SDM-79, in which promastigotes, after 6 days of culture, reached cell densities as high as 4 × 10⁷ cells/ml. Cells growing in LIT or MTL medium showed longer division times and lower cell densities. We succeeded in obtaining Phytomonas sp. amastigote and spheromastigote forms in modified GRACE's medium, yielding transformation rates of up to 70%. Electron microscopy studies were performed in order to characterize the ultrastructural features of these forms obtained in vitro. On the other hand, metabolic studies based on qualitative (nuclear magnetic resonance spectroscopy) and quantitative metabolic methods (enzymatic assays) showed that promastigote forms secreted mainly ethanol, acetate, glycine, glycerol, piruvate and succinate in SDM-79 medium, whereas the major metabolites found after transformation in modified Grace's medium were ethanol, acetate, glycine, piruvate and smaller amounts of glycerol.     

Studies on the effects of the antitumor agent camptothecin and derivatives on DNA. Camptothecin potentiated cleavage of DNA by bleomycin in vitro

Addition of sodium camptothecin (2a, Fig. 1) in comparable low concentrations to the glycopeptide antitumor antibiotic bleomycin (BLM) leads to enhanced rates of single-strand scission of PM2-covalently closed circular DNA, whereas sodium camptothecin alone has no effect. A similar enhancement of DNA scission by sodium camptothecin is produced with the 1 : 1 bleomycin-iron complex alone or in conjunction with NADPH as an additional reductant. The interpretation that camptothecin may substitute for the reducing requirement of the antibiotic is supported by its oxidation at 37°C by the 1 : 1 bleomycin iron complex, by iron salts or more efficiently by hydrogen peroxide to the known hemiacetal (3, Fig. 1).Electrochemical studies of 2a, its analogues and selected model compounds established that the α-pyridone ring D is most susceptible to a one-electron reduction at a reversible potential of ?0.95 ± 0.01 V. The reduced camptothecin is a transient species readily capable of donating an electron. This process may by compatible with a coupled reduction of the sequestered Fe(III) in the glycopeptide antibiotic necessary for the expression of antibiotic and antitumor properties. The results may provide a mechanistic rationale for the observed potentiation of the antitumor activity of bleomycin by camptothecin in vivo.     

Specific Expression of N-Acetylaspartate in Neurons, Oligodendrocyte-Type-2 Astrocyte Progenitors, and Immature Oligodendrocytes In Vitro

To test the specificity of N-acetylaspartate (NAA) as a neuronal marker for proton nuclear magnetic resonance (1H NMR) spectroscopy, purified and characterized cultured cells were analyzed for their NAA content using both 1H NMR and HPLC. Cell types studied included cerebellar granule neurons, type-1 astrocytes, meningeal cells, oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells, and oligodendrocytes. A high concentration of NAA was found in extracts of cerebellar granule neurons (approximately 12 nmol/mg of protein), whereas NAA remained undetectable in purified type-1 astrocytes, meningeal cells, and mature oligodendrocytes. However, twice the neuronal level of NAA was found in O-2A progenitors grown in vitro. In addition significant levels of NAA were also detected in cultures of immature oligodendrocytes. Our data partly support previous suggestions that NAA may be a useful neuronal marker for 1H NMR spectroscopic examination of the adult brain. However, they also raise the further possibility that alterations of NAA associated with some specific brain disorders, particularly disorders seen in newborn and young children, may reflect abnormalities in the development of oligodendroglia or their precursors.     

Intrinsically disordered inhibitor of glutamine synthetase is a functional protein with random‐coil‐like pKa values

The sequential action of glutamine synthetase (GS) and glutamate synthase (GOGAT) in cyanobacteria allows the incorporation of ammonium into carbon skeletons. In the cyanobacterium Synechocystis sp. PCC 6803, the activity of GS is modulated by the interaction with proteins, which include a 65‐residue‐long intrinsically disordered protein (IDP), the inactivating factor IF7. This interaction is regulated by the presence of charged residues in both IF7 and GS. To understand how charged amino acids can affect the binding of an IDP with its target and to provide clues on electrostatic interactions in disordered states of proteins, we measured the pK_a values of all IF7 acidic groups (Glu32, Glu36, Glu38, Asp40, Asp58, and Ser65, the backbone C‐terminus) at 100 mM NaCl concentration, by using NMR spectroscopy. We also obtained solution structures of IF7 through molecular dynamics simulation, validated them on the basis of previous experiments, and used them to obtain theoretical estimates of the pK_a values. Titration values for the two Asp and three Glu residues of IF7 were similar to those reported for random‐coil models, suggesting the lack of electrostatic interactions around these residues. Furthermore, our results suggest the presence of helical structure at the N‐terminus of the protein and of conformational changes at acidic pH values. The overall experimental and in silico findings suggest that local interactions and conformational equilibria do not play a role in determining the electrostatic features of the acidic residues of IF7.     

fMRI Hippocampal Activity During a VirtualRadial Arm Maze

Numerous studies have shown that the hippocampus is critical for spatial memory. Within nonhuman research, a task often used to assess spatial memory is the radial arm maze. Because of the spatial nature of this task, this maze is often used to assess the function of the hippocampus. Our goal was to extrapolate this task to humans and examine whether healthy undergraduates utilize their hippocampus while performing a virtual reality version of the radial arm maze task. Thirteen undergraduates performed a virtual radial arm maze during functional magnetic resonance imaging. The brain maps of activity reveal bilateral hippocampal BOLD signal changes during the performance of this task. However, paradoxically, this BOLD signal change decreases during the spatial memory component of the task. Additionally, we note frontal cortex activity reflective of working memory circuits. These data reveal that, as predicted by the rodent literature, the hippocampus is involved in performing the virtual radial arm maze in humans. Hence, this virtual reality version may be used to assess the integrity of hippocampus so as to predict risk or severity in a variety of psychiatric disorders.     

O‐glycoside sequence of pentacyclic triterpene saponins from Phytolacca bogotensis using HPLC‐ESI/multi‐stage tandem mass spectrometry

Introduction – The lack of pharmacopoeial methodologies for the quality control of plants used for therapeutic purposes is a huge problem that impacts directly upon public health. In the case of saponins, their great structural complexity, weak glycoside bonds and high polarity hinder their identification by conventional techniques. Objective – To apply high‐performance liquid chromatography–electrospray tandem mass spectrometry (HPLC‐ESI/MSⁿ) to identify the O‐glycoside sequence of saponins from the roots of Phytolacca bogotensis. Methodology – Saponins were isolated by preparative HPLC and characterised by NMR spectroscopic experiments. Collision‐induced dissociation (CID) of isolated saponins was performed producing typical degradation reactions that can be associated with several glycosidic bonds as empirical criteria. A method using solid‐phase extraction (SPE) and HPLC/ESI‐MSⁿ for the characterisation of saponins and identification of novel molecules is described. Results – Three saponins reported for the first time in P. bogotensis were isolated and characterised by NMR spectroscopy. Characteristic cross ring cleavage reactions have been used as empirical criteria for the characterisation of the glycosidic bonds most frequently reported for Phytolacca saponins. One new saponin was proposed on the basis of empirical criteria, and other five saponins were identified for the first time for P. bogotensis using HPLC‐ESI/MSⁿ. Conclusion – Electrospray ionisation in combination with tandem mass spectrometry has been established as a powerful tool for the profiling of saponins from roots of P. bogotensis. CID proved to be a useful tool for the characterisation and identification of known and novel saponins from the plant family Phytolaccaceae and can be used for quality control purposes of crude plant extracts. Copyright © 2009 John Wiley & Sons, Ltd.