去锌胰岛素与脂质小胞相互作用的核磁共振研究

用高分辨核磁共振技术,以脂质小胞为模型膜,研究膜与胰岛素分子之间的相互作用,处于胰岛素分子表面所包含B链的疏水面,很可能伸入膜脂质双层,对该双层产生微扰。中性pH范围是二者结合的最佳条件。     

New diphenylhexatriene derivatives as fluorescent membrane probes: Partitioning properties

Three new diphenylhexatriene derivatives, two phospholipids and one single-chain amphiphilic molecule, have been synthesized and considered as probes for measuring membrane fluidity by fluorescence anisotropy. The possibility of using these probes to determine specifically fluidity of inner leaflets of cellular plasma membranes was inferred from their partitioning properties between gel and liquid crystalline phases of phospholipid vesicles of binary composition.     

2D 1H-31P solid-state NMR studies of the dependence of inter-bilayer water dynamics on lipid headgroup structure and membrane peptides

The dynamics of hydration-water in several phospholipid membranes of different compositions is studied by 2D (1)H-(31)P heteronuclear correlation NMR under magic-angle spinning. By using a (1)H T(2) filter before and a (1)H mixing-time after the evolution period and (31)P detection, inter-bilayer water is selectively detected without resonance overlap from bulk water outside the multilamellar vesicles. Moreover the (1)H T(2) relaxation time of the inter-bilayer water is measured. Lipid membranes with labile protons either in the lipid headgroup or in sterols exhibit water-(31)P correlation peaks while membranes free of exchangeable protons do not, indicating that the mechanism for water-lipid correlation is chemical exchange followed by relayed magnetization transfer to (31)P. In the absence of membrane proteins, the inter-bilayer water (1)H T(2)'s are several tens of milliseconds. Incorporation of charged membrane peptides shortened this inter-bilayer water T(2) significantly. This T(2) reduction is attributed to the peptides' exchangeable protons, molecular motion and intermolecular hydrogen bonding, which affect the water dynamics and the chemically relayed magnetization transfer process.     

1H AND 31P NMR of Cat Optic Nerve

The ³¹P and ¹H NMR spectra of perchloric acid extracts of cat optic nerve were obtained. Nerves were exposed by lateral orbitotomy, frozen in situ with liquid nitrogen, removed, and extracted.

Resonances from the high energy phosphates adenosine triphosphate and phosphocreatine, and compounds of phospholipid metabolism, such as phosphorylcholine, phosphorylethanolamine, and their glyceryl esters, were observed and assigned in the ³¹P spectrum. The ³¹P spectrum of cat optic nerve ex vivo also was obtained. Resonances from numerous low-molecular-weight compounds including creatine, N-acetyl-aspartate (NAA), inositol, choline, and various amino acids were observed in the ¹H spectrum and assigned by comparison to the spectra of model compounds. The neurotransmitter N-acetylaspartylglutamate (NAAG), previously found in retinal ganglion cells and implicated in the transmission of visual information, was resolved from NAA and observed for the first time by NMR in tissue extracts at a level higher than that of NAA.     

蜂毒肽浓度和磷脂组分对巨囊泡泄露的影响

蜂毒肽作为一种广谱抗菌肽已经得到广泛认知,用蜂毒肽构建载药体系攻击癌细胞研究正在兴起.基于仿生物膜模型探索其破坏机理,可以避免潜在活性细胞过程的影响.在此,我们选用细胞尺寸的单层巨囊泡膜模型,可在光学显微镜下直接观察和操作,获得仿正常细胞膜和仿癌细胞膜在不同蜂毒肽浓度刺激下的响应.研究得出,低浓度蜂毒肽诱导囊泡泄露实验表明中性磷脂囊泡以孔模式为主泄露,负电性磷脂囊泡以爆裂模式为主泄露;高浓度蜂毒肽诱导囊泡泄露实验表明负电性磷脂相较于中性磷脂可延迟蜂毒肽作用效果;蜂毒肽色氨酸残基荧光光谱表明囊泡膜表面蜂毒肽吸附量以及泄露模式依赖于磷脂组分.此外,推断了蜂毒肽对不同组分磷脂膜的破坏作用模型.研究为蜂毒肽在肿瘤细胞的作用机制及其衍生物的优化设计提供参考.     

白血病人血清的31P NMR研究

运用³¹P核磁共振波谱(NMRS)研究血清中磷脂的代谢变化,并探讨该法对白血病诊断的可行性.方法采用MSL-300MHz型超导核磁共振仪测量白血病人及健康人血清标本,通过分析比较血清中磷脂酰胆碱(PC)信号及磷脂酰乙醇胺和(神经)鞘磷脂(PE+SM)信号相对积分面积,了解各自血清中的磷脂含量水平.结果,白血病人 血清中的磷脂信号强度相对正常人明显偏低,反映了对应的PC及(PE+SM)含量相对正常人偏低,尤其对初发未治疗的病人,差别更加明显.结论,本法可作为研究血清中磷脂的变化、代谢情况及白血病人临床诊断的新方法.     

Metabolite mapping of human filarial parasite, Brugia malayi with nuclear magnetic resonance

Metabolite mapping of human filarial parasite, Brugia malayi was carried out in vitro as well as in situ in host Mastomys coucha by ³¹P nuclear magnetic resonance (NMR) spectroscopy. Detection of parasites by visualizing contrast spots due to pathologic changes was observed by ¹H magnetic resonance imaging (MRI). Major metabolites of adult B. malayi observed by ³¹P-NMR spectroscopy were of sugar phosphates (SP), phosphomonoesters (PME), glycerophosphoryl-ethanolamine (GPE), -choline (GPC), phosphoenolpyruvate (PEP), inorganic phosphate (Pi), nucleoside diphosphosugar and nucleotides-mono, -di and -tri phosphates. PEP and GPC were present in high concentration; PEP being the major energy reservoir and GPC the major phospholipid in this species of filaria. The ³¹P NMR spectra of testis of mastomys, showed seven major peaks of SP, PME, phosphocreatine (PCr), phosphodiesters (PDE), Pi, and nucleotides di- and tri-phosphates. The ³¹P-NMR spectra of testis of B. malayi infected animal also consisted of seven major peaks with significant decrease in the SP and PME peak showing changes in the carbohydrate and lipid metabolism of filaria infected testis. Thus, in vivo ³¹P MRS provided a non-invasive assessment of tissue bioenergetics and phospholipid metabolism.     

Phosphorus-31 NMR studies of stabilized phosphorus ylids in the solid state.

Phosphorus-31 powder NMR spectra and high-resolution MAS spectra have been obtained for a number of stabilized phosphorus ylids under conditions of high-power proton decoupling and cross-polarization. The 31P CP/MAS spectra are compared to those obtained from isotropic solutions. The variation of chemical shift anisotropy and of the principal components of the 31P chemical shift tensor determined from 31P powder NMR line shapes are discussed in terms of the relative importance of accepted valence bond resonance structures. The results indicate that the invariance of the isotropic chemical shift, delta iso, observed in previous 31P NMR investigations of phosphorus ylids in solution is due to fortuitous cancellation of opposing changes in the principal components, delta 11 and delta 33, of the 31P chemical shift tensor. The 31P dipolar NMR powder spectrum of a typical stabilized ylid, (C6H5)3(31)P-13CHC(O)OCH2CH3, is analyzed in order to obtain the orientation of the 31P chemical shift tensor with respect to the 31P-13C alpha dipolar vector.     

Modeling of 31P-NMR spectra of magnetically oriented phospholipid liposomes: A new analytical solution

³¹P-NMR spectroscopy is widely used for studies of phospholipid liposomes, a commonly used model of a biological membrane. For the correct analysis of ³¹P-NMR spectra of the liposomes it is necessary to take into account that they are deformed by the magnetic field of the spectrometer. The liposomes become ellipsoidal and this affects the lineshape of the spectrum. In the present communication we suggest a new analytical formula for modeling of ³¹P-NMR spectra of the prolate phospholipid liposomes. The formula assumes a Lorentzian broadening function and exactly ellipsoidal shape of the liposomes. Based on the formula a program called P-FIT is designed for the practical analysis of the experimental multicomponent spectra of the prolate liposomes. The versatility of the program developed in a Mathematica environment is demonstrated by simulations of a number of ³¹P-NMR spectra with different complexity.     

Observation of rat hind limb skeletal muscle during arterial occlusion and reperfusion by 31P MRS and 1H MRI

31P NMR spectra and 1H MR T1- and T2-weighted spin-echo images were concurrently observed in rat hind limb during arterial occlusion and following reperfusion. With arterial occlusion, phosphocreatine level decreased and inorganic phosphate (Pi) level increased in 31P NMR spectra. Intracellular pH's dropped as a function of time. Beta-ATP started to decrease in three hours. In six hours after the occlusion, any peaks other than Pi were scarcely detected. The signal intensities in the 1H MR images increased homogeneously in both T1- and T2-weighted conditions, but the changes were more profound with T2-weighted images. After the release of the arterial occlusion, the 31P NMR spectra recovered to the preischemic state in several hours. The 1H MR images during reperfusion showed characteristic heterogenous pattern. The signal intensities in the anterior tibial muscle and the gastrocnemius muscle remained high in T1-weighted condition and the intensities further increased in T2-weighted condition, while those in other parts returned to the preischemic level. These changes were found to be irreversible even 12 hr after the release. The high signal intensities suggested the increase of water in the extracellular compartment induced by so-called reperfusion injury. Multinuclear analysis using in vivo NMR was valuable to consecutively detect time-dependent and location-specific response in skeletal muscle during ischemia and reperfusion.     

AssignFit: a program for simultaneous assignment and structure refinement from solid-state NMR spectra

AssignFit is a computer program developed within the XPLOR-NIH package for the assignment of dipolar coupling (DC) and chemical shift anisotropy (CSA) restraints derived from the solid-state NMR spectra of protein samples with uniaxial order. The method is based on minimizing the difference between experimentally observed solid-state NMR spectra and the frequencies back calculated from a structural model. Starting with a structural model and a set of DC and CSA restraints grouped only by amino acid type, as would be obtained by selective isotopic labeling, AssignFit generates all of the possible assignment permutations and calculates the corresponding atomic coordinates oriented in the alignment frame, together with the associated set of NMR frequencies, which are then compared with the experimental data for best fit. Incorporation of AssignFit in a simulated annealing refinement cycle provides an approach for simultaneous assignment and structure refinement (SASR) of proteins from solid-state NMR orientation restraints. The methods are demonstrated with data from two integral membrane proteins, one α-helical and one β-barrel, embedded in phospholipid bilayer membranes.     

Assigning solid-state NMR spectra of aligned proteins using isotropic chemical shifts

A method for assigning solid-state NMR spectra of membrane proteins aligned in phospholipid bicelles that makes use of isotropic chemical shift frequencies and assignments is demonstrated. The resonance assignments are based on comparisons of 15N chemical shift differences in spectra obtained from samples with their bilayer normals aligned perpendicular and parallel to the direction of the applied magnetic field.     

直肠癌及直肠正常组织的IR和~1H及~(31)P NMR谱研究

对12例直肠组织的红外光谱图进行了测定,并对6例直肠癌变及正常组织的固体1H和31P NMR谱与另外6例直肠癌变及正常组织的液体1H和31P-NMR谱进行研究.结果表明,(1)直肠癌变和正常组织的红外光谱中存在明显差异.(2)在固体1H和31P NMR谱中,1H NMR谱上癌组织的脂肪酸与水相关的特征峰面积的比值I(5+6+7)/I(1+2+3+4)明显变低,依据I(5+6+7)/I(1+2+3+4)比值可区分癌变与正常组织.31P NMR谱上,癌变组织的最强峰的化学位移明显向高场偏移.(3)在液体1H和31P NMR谱中,1H NMR谱上癌组织中3.20处峰强较正常组织高,表明与癌变密切相关的胆碱类化合物包括游离的胆碱、磷酸胆碱及甘油磷酸胆碱等的量增大.31P NMR谱上,癌变组织的强度最大峰的化学位移分别为1.18,1.26,1.29,1.37,正常组织的化学位移为1.70和2.18,癌变组织的强度最大峰的化学位移明显向高场偏移.通过癌变与正常组织代谢物1H NMR谱峰强度和1P NMR谱峰化学位移的差异,可以区分直肠癌变和正常组织,核磁共振1H和31P NMR谱技术可能发展成为一种诊断直肠癌的新方法.     

Interaction of keratolytic drug,salicylic acid with dipalmitoyl phosphatidylethanolamine vesicles

Salicylic acid (SA), a keratolytic drug, is used to treat skin disorder like corns, warts, and acne. To understand the mechanism by which SA interacts with the cell membranes, we have investigated its interaction with dipalmitoyl phosphatidylethanolamine (DPPE) vesicles using DSC, ¹H NMR and Raman spectroscopy. Presence of drug asymmetrically broadened the acyl chain melting transition and shifted the transition temperature, T _m, to lower value. Both, NMR and DSC studies indicate that the drug molecules are located in the glycerol backbone region of the lipid bilayer and increase the membrane headgroup fluidity. At high drug concentration, additional transitions are observed whose intensity increases with increasing drug concentration. In cholesterol doped DPPE dispersion the interaction of SA with DPPE bilayer is more. The transformation of the gel phase of DPPE dispersion to a stable crystalline subgel phase(s) is accelerated by the presence of SA.     

Influence of salicylic acid on the biophysical properties of dipalmitoyl phosphatidylcholine vesicles

The effect of the keratolytic drug salicylic acid (SA) on the thermotropic behaviour, and dynamics of dipalmitoyl phosphatidyl choline (DPPC)–water/buffer pH?7.4 vesicles was studied using DSC and ¹H NMR. In both systems, incorporation of SA in DPPC bilayer causes a significant depression in the transition temperature of both the pre-transition (PT) and the gel-to-liquid crystalline (CM) transition. The presence of the drug reduces the cooperativity of both the PT and CM transitions. These findings indicate that SA is bound strongly to the lipid bilayer leading to increased membrane fluidity. The DPPC vesicles incorporated with high drug concentration show phase segregation. One of the interesting findings in this study is the formation of a more ordered high temperature gel (L_β2) phase when the SA-doped DPPC dispersion is prepared at physiological pH. The effect of inclusion of cholesterol in the SA-free and SA-doped DPPC dispersion was also studied.     

A Spectroscopic Study of the Interaction of the Fluorescent β-Carboline-3-carboxylic Acid N-methylamide with DNA Constituents: Nucleobases, Nucleosides and Nucleotides

Interaction between beta-carboline-3-carboxylic acid N-methylamide, betaCMAM, and nucleobases, nucleosides and nucleotides is studied in the ground state with UV-visible, (1)H NMR and (31)P NMR spectroscopies and in the first excited state, with steady-state and time-resolved fluorescence spectroscopy. Job plots show a predominant 1:1 interaction in both electronic states. Association constants are estimated from changes in the absorption spectra, and show that the strongest interaction is produced with the nucleosides: 2'-deoxyadenosine (dAdo) and thymidine (Thd), and with the mononucleotides: 2'-deoxycytidine 5'- monophosphate (5'-dCMP) and uridine 5'- monophosphate (5'-UMP). These results are corroborated by the upfield shifts of two (1)H NMR resonances of the betaCMAM indole group. The (31)P NMR resonance of nucleotides is shifted downfield, suggesting the presence of electrostatic or hydrogen bond interaction with betaCMAM. In the first electronic singlet excited state, static and dynamic quenching of betaCMAM emission is achieved upon addition of nucleobases, nucleosides and nucleotides. This has been analysed using Stern-Volmer kinetics.     

Effects of N-Phosphoproline on the Phospholipid of Human Erythrocytes Membrane

The effects of phosphorylated proline on the packing and conformation of the membrane phospholipid have been studied. Generally it causes the polar parts of the phospholipid to become more mobile, the packing of the long chain of phospholipid to become more loose, the long chain to be in a lower trans/gauche ratio, and the end of the long chain to become more orderly. Any change in the level of phosphorylated proline affects the fluidity of the membrane phospholipid. Free proline and phosphorylated proline have different effects on the aliphatic chains of phospholipid.     

Combined 17O NMR and 11B-31P double resonance NMR studies of sodium borophosphate glasses

17O enriched sodium borophosphate glasses were prepared from isotopically enriched NaPO3 and H3BO3. These glasses have been studied by 17O, 11B and 31P NMR including 17O and 11B multiple quantum magic angle sample spinning (MQMAS), 11B-31P heteronuclear correlation (HETCOR) NMR and 11B{31P} rotational echo double resonance (REDOR). For comparison, the crystalline borophosphates BPO4 and Na5B2P3O13 were included in the investigations. The latter compound shows three sharp 31P resonances at -0.2, -2 and -8 ppm and two BO4 sites that can only be resolved by MQMAS. The 17O NMR spectra were recorded using both the static echo method at medium magnetic field (9.4 T) as well as MAS and MQMAS methods at high field (17.6 T). In total, five oxygen sites were identified in these borophosphate glasses: P-O-P, Na...O-P, P-O-B, B-O-B, Na...O-B. However, these five sites are not present simultaneously in any of the glasses. The 17O MQMAS spectra prove that P-O-B links play a major role in borophosphate glasses. These results are confirmed by the complementary 11B MAS spectra that show the presence of asymmetric and symmetric trigonal groups BO3a and BO3s and two tetrahedral BO4 units. 11B{31P} REDOR NMR is used to give independent information to assign the 11B lines to structural units present in the glasses. These REDOR measurements reveal that B-O-P bonds are present for each borate unit, including the BO3 groups. Particularly, a structural proposal for the two different BO4 resonances is given in terms of a different number of bonded phosphate tetrahedra. The 31P MAS spectra are usually broad and not well resolved. It is shown by 11B-31P HETCOR NMR that a possible structural assignment of a 31P signal at about -20 ppm to Q2 units as in binary sodium phosphate glasses is wrong and that the phosphate tetrahedron belonging to this resonance must be connected to borate groups.     

应用圆二色光谱研究交变应力对烟草细胞膜蛋白结构的影响

本文用圆二色光谱(CD)研究了交变应力对烟草细胞膜蛋白结构的影响 采用本实验室研制的强声波发生装置来产生交变应力场,研究了不同强度和频率的交变应力场作用后烟草细胞膜蛋白结构的变化 研究结果表明:细胞膜蛋白结构变化与应力的频率和强度密切相关,一定频率和强度范围内的交变应力能使得烟草细胞膜蛋白的二级结构发生明显的变化,表现为α螺旋的增加和 β转角的减少 这反映了细胞膜蛋白和磷脂相互作用的增强和细胞膜流动性的增加,进而为细胞的生长和分裂提供有利的条件 从细胞和分子水平研究交变应力对植物生长、发育的影响及其作用机制是一个很有意义的尝试.     

Phosphorus-31 MAS NMR investigations of pyridine stabilized dithiomonometaphosphoryl halides

Phosphorus-31 magic-angle spinning NMR spectroscopy was chosen in order to study a series of N-donor stabilized dithiomonometaphosphoryl halides (F, Cl, and Br) and to extract structural and bonding information from their spectra. Additionally, an unusual line splitting was observed in the 31P MAS NMR spectra of pyridine dithiomonometaphosphoryl chloride (Py-->PS2Cl) and the analogous bromide (Py-->PS2Br). It is shown that this effect is due to a residual dipolar coupling between phosphorus and chlorine or bromine, respectively. This work is to our knowledge the first observation of this effect between P and Br in 31P MAS NMR spectra.