NMR observation of Tau in Xenopus oocytes

The observation by NMR spectroscopy of microinjected 15N-labelled proteins into Xenopus laevis oocytes might open the way to link structural and cellular biology. We show here that embedding the oocytes into a 20% Ficoll solution maintains their structural integrity over extended periods of time, allowing for the detection of nearly physiological protein concentrations. We use these novel conditions to study the neuronal Tau protein inside the oocytes. Spectral reproducibility and careful comparison of the spectra of Tau before and after cell homogenization is presented. When injecting Tau protein into immature oocytes, we show that both its microtubule association and different phosphorylation events can be detected.     

Atomic force microscopy of living and fixed Xenopus laevis embryos

Xenopus laevis embryos are a rather simple and at the same time a very interesting animal model, which is widely used for research in developmental biology. Intensive coordinated cell movements take place during the multi-cellular organism development. Little is known of the cellular, molecular and biomechanical mechanisms of these movements. The conceptual framework for analysis of cell interactions within integrated populations is poorly developed. We have used atomic force microscopy (AFM) to observe the surface of fixed X. laevis embryos at different stages of their development. We have developed a new sample preparation protocol for these observations. The obtained images were compared with scanning electronic microscopy (SEM) data. Cell rearrangement during morphogenesis in vivo was also visualized by AFM. In the current paper we discuss facilities and challenges of using this technique for further embryo researching.     

Evaluation of spin labels for in-cell EPR by analysis of nitroxide reduction in cell extract of Xenopus laevis oocytes

Spin-label electron paramagnetic resonance (SL-EPR) spectroscopy has become a powerful and useful tool for studying structure and dynamics of biomacromolecules. However, utilizing these methods at physiological temperatures for in-cell studies is hampered by reduction of the nitroxide spin labels and thus short half-lives in the cellular environment. Consequently, reduction kinetics of two structurally different nitroxides was investigated in cell extracts of Xenopus laevis oocytes using rapid-scan cw-experiments at X-band. The five member heterocyclic ring nitroxide PCA (3-carboxy-2,2,5,5-tetramethylpyrrolidinyl-1-oxy) under investigation features much higher stability against intracellular reduction than the six member ring analog TOAC (2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxilic acid) and is therefore a suitable spin label type for in-cell EPR. The kinetic data can be described according to the Michaelis–Menten model and thus suggest an enzymatic or enzyme-mediated reduction process.     

Estimation of pore sizes in porous silicon by scanning electron microscopy and NMR cryoporometry

Certain possibilities of scanning electron microscopy and cryoporometry based on nuclear magnetic resonance (NMR) have been evaluated to determine pore sizes in porous silicon. The results obtained by these methods have been compared. NMR cryoporometry has been shown to be promising in the investigation of porous materials.     

NMR investigation of water and methanol mobility in nanocomposite fuel cell membranes

Water and methanol transport behavior, morphology, and solvent adsorption of filler-free Nafion membrane, Nafion–SiO₂, Nafion–TiO₂, and two Nafion–Zr(HPO₄)₂ composites were investigated using nuclear magnetic resonance methods, including spin-lattice relaxation and pulsed-field-gradient spin-echo diffusion conducted under both variable temperature and variable hydrostatic pressure conditions and scanning electron microscopy analysis. A comparison between water and methanol self-diffusion coefficients reveals that the water mobility is higher than the methanol mobility in all the membranes. Additionally, the inclusion of inorganic fillers improves both the solvent uptakes and the transport properties of the composite membranes relative to filler-free Nafion, with the exception of one of the Nafion–Zr(HPO₄)₂ composite. Nafion–Zr(HPO₄)₂ composites were prepared by two different procedures, in situ and ex situ. Although phosphorus-31 magic-angle spinning nuclear magnetic resonance spectra show the same structures of the particles in both kinds of membranes, the morphology, solvent absorption properties, and solvent mobilities are very different. Paper presented at the 11th EuroConference on the Science and Technology of Ionics, Batz-sur Mer, Sept. 9–15, 2007.     

Transdermal water mobility in the presence of electrical fields using MR microscopy.

Magnetic resonance microscopy of skin from hairless rats under the influence of electrical fields was conducted for two cases: 1) low voltage constant electrical fields and 2) high-voltage short pulse, electrical fields. Under conditions of the low voltage and low current iontophoresis, i.e., 0 to 20 V, and 0 to 0.5 mA/cm2, it was found that the skin structure, as observed by magnetic resonance microscopy, did not significantly change until 20 Volts were applied across the 0.1 cm thick skin. Under these conditions, the viable epidermis appeared to swell, and this result corresponded to observations from scanning electron microscopy and other research from the literature. High voltage electrical fields, i.e., 220 V 1 ms pulses repeated once per second, appeared to hydrate the stratum corneum as is consistent with published literature on electroporation. In the case of iontophoresis, water self-diffusion coefficients in the epidermis and hair follicle regions at all voltages were affected by the electrical field. Statistical analysis at the 95% confidence level for the comparison of the average differences between diffusion coefficients with the electrical field on and with the electrical field off for pair matched pixels for the viable epidermis show that for 5 V (p = 0.00377), 10 V (p = 0.0108), 20 V (p = 0.0219) regimes there are statistically significant (p < or = 0.05) changes due to the applied electric field. The same analysis for the hair follicle region at 5 V (p = 6.89 x 10(-7)), 10 V (p = 1.42 x 10(-5)), 20 V (p = 3.23 x 10(-3)) also show statistically significant changes (p < or = 0.05). When the electroporation pulse was applied, the water diffusion coefficients increased by about 30% to 6.6 x 10(-6) cm2/s +/- 2.4 x 10(-7) cm2/s and 8.3 x 10(-6) cm2/s +/- 3.7 x 10(-7) cm2/s, for the epidermis and hair follicle regions, respectively. Significant differences were noted between diffusion coefficients in the viable epidermis and the hair follicles for all cases.     

In vivo quantitation of water content in muscle tissues by NMR imaging.

The water contents of phantoms and muscle tissues were determined directly from NMR imaging experiments. The method involves the calculation of corrected proton densities using relaxation time determinations and suitable calibration phantoms. Comparison with the values obtained from the oven-dry method yields good agreement in normal rat skeletal tissue and in rats injected with red blood cells from sickle cell patients.     

Molecular mobility in fixed-bed reactors investigated by multiscale NMR techniques

The complex problem of a fixed-bed reactor consisting of catalytically active particles provides an exceptional opportunity of combining a wide range of NMR methods which have become available over time as tools to probe porous media. This work demonstrates the feasibility of different NMR techniques for the investigation of the intra- and interparticle pore space over length scales from nanometers up to centimeters. Many industrially relevant cracking reactions leave a coke residue on the inner surface of the porous catalyst particles so that the active sites become inaccessible to the reactants. Moreover, the pore space shrinks due to the formation of coke, thereby hindering molecular transport. The presence of the coke residue and its influence on the mobility of adsorbed fluid molecules are probed by 129Xe spectroscopy, NMR cryoporometry, relaxation dispersion measurements, and investigations of the reduced diffusivity in the intraporous space. The voids surrounding the random arrangement of catalyst pellets represent another pore space of much larger dimensions, the properties of which can be more directly investigated by mapping the fluid density and the velocity distribution from velocity-encoded imaging. Propagator representations averaged over large sample volumes are discussed and compared to velocity images obtained in selected axial slices of the reactor.     

Imaging the velocity profiles in tubeless siphon flow by NMR microscopy

We report on the use of NMR micro-imaging to observe flow within a tubeless siphon. The flow is maintained in a visco-elastic liquid of high extensional viscosity, namely 0.5% w/v 8 million Dalton polyethylene oxide in water. The velocity profiles reveal a significant velocity gradient in the vertical direction as well as a transition from near-Poiseuille flow at the pipe entrance to plug flow far from the pipe entrance towards the base of the tubeless siphon.     

Measurement of the maximum content of bound water in bentonitic clay using dielectric and NMR methods

An investigation of moisture dependences of refractive index and spectral width of the nuclear magnetic resonance (NMR) is performed for bentonitic clay specimens at different temperatures. These moisture dependences allowed identifying two types of moisture: bound and free water. The maximum content of water in the bentonitic clay is measured.     

融合冠层水分特征的光谱参数NCVI及反演玉米LAI

精确反演农作物冠层叶面积指数对指导作物管理和作物估产具有非常重要的意义。以吉林市郊区玉米种植区为试点,考虑冠层叶片水分含量对LAI的贡献,在NDVI的基础上结合表征冠层叶片水分含量的植被指数DSWI,提出一种归一化综合植被指数NCVI,以此建立模型反演LAI,并对模型进行检验。结果表明:NCVI模型反演LAI值与实测值之间存在良好的对应关系,此模型突破了传统经验模型对稠密冠层LAI反演的局限,对LAI值大于3的冠层反演效果良好;另外,NCVI模型对土壤水环境十分敏感,在干旱半干旱地区的反演效果明显优于一般区域。     

Velocity and diffusion imaging in dynamic NMR microscopy

The imposition of resolution gradients in a pulsed-gradient spin-echo (PGSE) NMR sequence induces motionally dependent phase and amplitude modulation in the image, a technique which we have termed dynamic NMR microscopy. Fourier analysis of this modulation gives a dynamic displacement profile for each pixel which can then be analyzed to obtain velocity and diffusion maps. The application of this method at high spatial resolution is motivated by a desire to measure vascular flow in living plants and variations in molecular self-diffusion under the influence of velocity shear in narrow capillaries. The theory of dynamic NMR microscopy is presented and potential artifacts discussed, including the effect of slice selection gradients, PGSE gradient nonuniformity, and specific problems associated with the measurement of self-diffusion in the presence of velocity gradients. It is demonstrated that a double-echo PGSE pulse sequence can be used to restore coherent phase shifts associated with steady-state flow, and examples of self-diffusion maps and signed velocity maps from sequences of phase-encoded images obtained by projection reconstruction are given. This method has been applied at 20,um transverse resolution in laminar capillary flow.     

The influence of water content on the 23Na and 31P NMR spectral parameters in solid Na-DNA

23Na and 31P MAS NMR spectra and spin-lattice relaxation times in a solid sample of Na-DNA were measured under very carefully controlled conditions of relative ambient humidity. The observed substantial changes of the NMR parameters are related to the water-induced transitions between the different molecular configurations of DNA, with the transition between A- and B-DNA occurring in the 30-60% range of relative humidity. Our work demonstrates that the previously measured NMR parameters are in error because the relative humidity of the system had not been controlled, rendering the results irreproducible.     

Estimation of relative water content in rice panicle based on hyperspectral vegetation indexes under water saving irrigation

Determining grain water content is a traditional method to quantify maturity, but this method is laborious. Ground-based remote sensing with rapidness and flexibility has been widely used in evaluating rice growth, but less attention has been paid to predicting physiological maturity. A field experiment with rice was conducted to investigate the effects of water saving irrigation on physiological maturity based on hyperspectral data. The results indicated that, with higher coefficients of determination (R² = 0.93 and 0.87, respectively), higher residual prediction deviation (RPD = 3.54 and 2.58, respectively) and lower root mean square error (RMSE = 2.88 and 4.43, respectively) among the tested models, R₁₆₅₄/R₆₆₂ and R₅₄₆/R₅₆₂ were suggested as the optimal indexes for monitoring relative water content in rice panicle under flooding and wetting conditions, respectively. This finding is helpful in providing reference for monitoring rice physiological maturity.     

Estimation of diffusion coefficient by photoemission electron microscopy in ion-implanted nanostructures

We have fabricated parallel stripes of nanostructures in an n-type Si substrate by implanting 30 keV Ga⁺ ions from a focused ion beam (FIB) source. Two sets of implantation were carried out. In one case, during implantation the substrate was held at room temperature and in the other case at 400 °C. Photoemission electron microscopy (PEEM) was carried out on these samples. The implanted parallel stripes, each with a nominal dimension of 4000 nm × 100 nm, appear as bright regions in the PEEM image. Line scans of the intensities from the PEEM image were recorded along and across these stripes. The intensity profile at the edges of a line scan is broader for the implantation carried out at 400 °C compared to room temperature. From the analysis of this intensity profile, the lateral diffusion coefficient of Ga in silicon was estimated assuming that the PEEM intensity is proportional to Ga concentration. The diffusion coefficient at 400 °C has been estimated to be ∼1.3 × 10⁻¹⁵ m²/s. Across the stripes an asymmetric diffusion profile has been observed, which has been related to the sequence of implantation of these stripes and the associated defect distribution due to lateral straggling of the implanted ions.     

Osmotic and aging effects in caviar oocytes throughout water and lipid changes assessed by 1H NMR T1 and T2 relaxation and MRI

By combining NMR relaxation spectroscopy and magnetic resonance imaging techniques, unsalted (us) and salted (s) caviar (Acipenser transmontanus) oocytes were characterized over a storage period of up to 90 days. The aging and the salting effects on the two major cell constituents, water and lipids, were separately assessed. T1 and T2 decays were interpreted by assuming a two-site exchange model. At Day 0, two water compartments that were not in fast exchange were identified by the T1 relaxation measurements on the us oocytes. In the s samples, T1 decay was monoexponential. During the time of storage, an increment of the free water amount was found for the us oocytes, ascribed to an increased metabolism. T1 and T2 of the s oocytes shortened as a consequence of the osmotic stress produced by salting. Selective images showed the presence of water endowed with different regional mobility that severely changed during the storage. Lipid T1 relaxation decays collected on us and s samples were found to be biexponential, and the T1 values lengthened during storage. In us and s oocytes, the increased lipid mobility with the storage was ascribed to lipolysis. Selective images of us samples showed lipids that were confined to the cytoplasm for up to 60 days of storage.     

Determination of the physical network of entanglements and the molecular mobility in linear flexible polymers by the NMR method

In the present paper, a theory of free induction decay in linear flexible chain polymers is developed. Theoretical and experimental dependences of the spin-spin relaxation time T ₂ on the average molecular weight M _w and temperature are compared. It is shown that, with increasing M _w , the topological structure of linear polymers changes, and at M _w > 10⁵, a physical network entanglements (quasi-network) is formed. A correlation function of molecular motions is obtained, which indicates whether the quasi-network is formed in polymer melts.     

A novel application of NMR microscopy: measurement of water diffusion inside bioadhesive bonds.

The self-diffusion coefficient of water (D) inside bioadhesive bonds formed by dry and prehydrated hydrophilic matrices has been spatially resolved using nuclear magnetic resonance (NMR) microscopy. One-dimensional profiles showing the variation of D inside bioadhesive bonds were calculated from nine diffusion-weighted profiles obtained immediately after bond formation and every 5 min for 30 min. The resulting data indicated that the hydration state of a hydrophilic matrix can significantly and dramatically influence the dynamics of water movement inside a bioadhesive bond.