Data processing of 3D and 4D in-vivo electron paramagnetic resonance imaging co-registered with ultrasound. 3D printing as a registration tool.

We present the concept of image registration using ultrasound (US) and electron paramagnetic resonance (EPR) imaging and discuss the benefits of this solution, as well as its limitations. Both phantoms and murine tumors were used to test US and EPR image co-registration. Comparison of dental molding cast immobilization and predesigned cradle revealed that the latter approach is more effective in stabilizing the fiducial position. In vivo imaging of mouse tumors, image registration and comparison of fiducials system for 3D spatial as well as 4D spatial-spectral EPR imaging supported by 3D US were demonstrated. Ultrasound may provide a convenient alternative to other anatomical imaging methods for image registration in preclinical research. Of particular interest is a fusion of US tissue structure, doppler vascular function and EPR oxygen or redox imaging.

Development of a PET/EPRI combined imaging system for assessing tumor hypoxia.

Precise quantitative delineation of tumor hypoxia is essential in radiation therapy treatment planning to improve the treatment efficacy by targeting hypoxic sub-volumes. We developed a combined imaging system of positron emission tomography (PET) and electron para-magnetic resonance imaging (EPRI) of molecular oxygen to investigate the accuracy of PET imaging in assessing tumor hypoxia. The PET/EPRI combined imaging system aims to use EPRI to precisely measure the oxygen partial pressure in tissues. This will evaluate the validity of PET hypoxic tumor imaging by (near) simultaneously acquired EPRI as ground truth. The combined imaging system was constructed by integrating a small animal PET scanner (inner ring diameter 62 mm and axial field of view 25.6 mm) and an EPRI subsystem (field strength 25 mT and resonant frequency 700 MHz). The compatibility between the PET and EPRI subsystems were tested with both phantom and animal imaging. Hypoxic imaging on a tumor mouse model using 18F-fluoromisonidazole radio-tracer was conducted with the developed PET/EPRI system. We report the development and initial imaging results obtained from the PET/EPRI combined imaging system.

Cytochrome a3: destruction by light.

Spectroscopic measurements on cultures of Prototheca zopfii irradiated with blue light revealed that inhibition of respiration was accompanied by destruction of cytochrome a(3). One of the three b-type cytochromes and one of the two c-type cytochromes of this organism were also affected. Cytochrome oxidase of yeast (not resolved into the a and a(3) components) and cytochrome a(3) of beef-heart mitochondria were also destroyed by blue light.

Interaction of tobamovirus movement proteins with the plant cytoskeleton.

The movement protein of tobacco mosaic tobamovirus and related viruses is essential for the cell-to-cell spread of infection and, in part, determines the host range of the virus. Movement protein (MP) was fused with the jellyfish green fluorescent protein (GFP), and a modified virus that contained this MP:GFP fusion protein retained infectivity. In protoplasts and leaf tissues, the MP:GFP fusion protein was detected as long filaments shortly after infection. Double-labeling fluorescence microscopy suggests that the MP interacts and coaligns with microtubules. The distribution of the MP is disrupted by treatments that disrupt microtubules, but not by cytochalasin B, which disrupts filamentous F-actin. Microtubules may target the MP to plasmodesmata, the intercellular channels that connect adjacent cells.

Plasmodesmata and plant cytoskeleton.

Plant cell-to-cell communication is achieved by membranous conduits called plasmodesmata, which bridge the cytoplasm of neighboring cells. A growing body of immunolocalization data shows an association of the cytoskeleton machinery with plasmodesmata. The role of the cytoskeleton in the plasmodesmata-mediated transport has been well documented for virus movement. Because viruses are known to exploit existing host pathways and because the cytoskeleton is involved in intracellular trafficking, the cytoskeleton is thought to drive and target macromolecules to plasmodesmata. It is this link between plasmodesmata and the cytoskeleton that will be described here.

A dysfunctional movement protein of tobacco mosaic virus interferes with targeting of wild-type movement protein to microtubules.

The Tobacco mosaic virus (TMV) movement protein (MPTMV) mediates cell-to-cell viral trafficking by altering properties of the plasmodesmata (Pd) in infected cells. During the infection cycle, MPTMV becomes transiently associated with endomembranes, microfilaments, and microtubules (MT). It has been shown that the cell-to-cell spread of TMV is reduced in plants expressing the dysfunctional MP mutant MPNT-1. To expand our understanding of the MP function, we analyzed events occurring during the intracellular and intercellular targeting of MPTMV and MPNT-1 when expressed as a fusion protein to green fluorescent protein (GFP), either by biolistic bombardment in a viral-free system or from a recombinant virus. The accumulation of MPTMV:GFP, when expressed in a viral-free system, is similar to MPTMV:GFP in TMV-infected tissues. Pd localization and cell-to-cell spread are late events, occurring only after accumulation of MP:GFP in aggregate bodies and on MT in the target cell. MPNT-1:GFP localizes to MT but does not target to Pd nor does it move cell to cell. The spread of transiently expressed MPTMV:GFP in leaves of transgenic plants that produce MPNT-1 is reduced, and targeting of the MPTMV:GFP to the cytoskeleton is inhibited. Although MPTMV:GFP targets to the Pd in these plants, it is partially impaired for movement. It has been suggested that MPNT-1 interferes with host-dependent processes that occur during the intracellular targeting program that makes MP movement competent.

Plant virus movement protein dynamics probed with a GFP-protein fusion.

A genetic fusion between the gene encoding green fluorescent protein (GFP) from the jellyfish Aequorea victoria, with that of the Ob-tobamovirus movement protein (MP) resulted in the expression of a fluorescent fusion protein (MP::GFP) that was fully biologically active in mediating the cell-to-cell spread of the Ob-virus. The MP::GFP fusion was used to follow in planta the subcellular trafficking of MP. GFP-tagged MP was transiently expressed and found to be associated with several subcellular compartments and structures including trans-wall structures, presumably plasmodesmata, and filament structures. The MP::GFP fusion can be used to monitor MP association with host proteins and structures, and for the isolation of interacting host components.

2D TRIPLE in orientationally disordered samples--a means to resolve and determine relative orientation of hyperfine tensors.

The two-dimensional (2D) TRIPLE experiment provides correlations between electron-nuclear double resonance (ENDOR) frequencies that belong to the same electron-spin manifold, M(S), and therefore allows to assign ENDOR lines to their specific paramagnetic centers and M(S) manifolds. This, in turn, also provides the relative signs of the hyperfine couplings. So far this experiment has been applied only to single crystals, where the cross-peaks in the 2D spectrum are well resolved with regular shapes. Here we introduce the application of the 2D TRIPLE experiment to orientationally disordered systems, where it can resolve overlapping powder patterns. Moreover, analysis of the shape of the cross-peaks shows that it is highly dependent on the relative orientation of the hyperfine tensors of the two nuclei contributing to this particular peak. This is done initially through a series of simulations and then demonstrated experimentally at a high field (W-band, 95 GHz). The first example concerned the (1)H hyperfine tensors of the stable radical alpha,gamma-bisdiphenylene-beta-phenylallyl (BDPA) immobilized in a polystyrene matrix. Then, the experiment was applied to a more complex system, a frozen solution of Cu(II)-bis(2,2':6',2'' terpyridine) complex. There, the 2D TRIPLE experiment was combined with the variable mixing time (VMT) ENDOR experiment, which determined the absolute sign of the hyperfine couplings involved, and orientation selective ENDOR experiments. Analysis of the three experiments gave the hyperfine tensors of a few coupled protons.

Improving W-band pulsed ENDOR sensitivity--random acquisition and pulsed special TRIPLE.

Two approaches for improving the signal-to-noise ratio (S/N) of W-band pulsed electron-nuclear double resonance (ENDOR) spectra are presented. One eliminates base-line problems while the other enhances the ENDOR effect. High field ENDOR spectra measured at low temperatures often suffer from highly distorted base-lines due to the heating effect of the RF pulses that causes some detuning of the cavity and therefore leads to a reduction in the echo intensity. This is a severe problem because it often masks broad and weak ENDOR signals. We show that it can be eliminated by recording the ENDOR spectrum in a random, rather than the standard sequential variation of the RF frequency. The S/N of the ENDOR spectrum can be significantly enhanced by the application of the pulse analog of the continuous wave (CW) special TRIPLE experiment. While this experiment is not applicable in the solid state at conventional X-band frequencies, at W-band it is most efficient. We demonstrate the efficiency of the special TRIPLE Davies and Mims experiments on single crystals and orientationally disordered systems.

The effect of spin relaxation on ENDOR spectra recorded at high magnetic fields and low temperatures.

A simple theoretical model that describes the pulsed Davies electron-nuclear double resonance (ENDOR) experiment for an electron spin S = (1/2) coupled to a nuclear spin I = (1/2) was developed to account for unusual W-band (95 GHz) ENDOR effects observed at low temperatures. This model takes into account the thermal polarization along with all internal relaxation processes in a four-level system represented by the electron- and nuclear-spin relaxation times T(1e) and T(1n), respectively, and the cross-relaxation time, T(1x). It is shown that under conditions of sufficiently high thermal spin polarization, nuclei can exhibit asymmetric ENDOR spectra in two cases: the first when t(mix) >> T(1e) and T(1n), T(1x) >> T(1e), where ENDOR signals from the alpha manifold are negative and those of the beta manifold positive, and the second when the cross- and/or nuclear-relaxation times are longer than the repetition time (t(mix) << T(1e) << t(R) and T(1n), T(1x) > t(R)). In that case the polarization of the ENDOR signals becomes opposite to the previous case, the lines in the alpha manifolds are positive, and those of the beta manifold are negative. This case is more likely to be encountered experimentally because it does not require a very long mixing time and is a consequence of the saturation of the nuclear transitions. Using this model the experimental t(mix) and t(R) dependencies of the W-band (1)H ENDOR amplitudes of [Cu(imidazole)(4)]Cl(2) were reproduced and the values of T(1e) and T(1x) >> T(1e) were determined. The presence of asymmetry in the ENDOR spectrum is useful as it directly provides the sign of the hyperfine coupling. The presented model allows the experimentalist to adjust experimental parameters, such as t(mix) and t(R), in order to optimize the desired appearance of the spectrum.

Studies on the longitudinal and lateral transport of IAA in the shoots of etiolated corn seedlings.

The auxin, indole-3-acetic acid, and the symplastic probe, carboxyfluorescein diacetate, were applied to the cut mesocotyl base or coleoptile apex of etiolated Zea mays seedlings and their transport measured and tissue distribution determined. The longitudinal transport of indole-3-acetate was strongly basipolar, while that of carboxyfluorescein was essentially apolar. The longitudinal transport of IAA, like carboxyfluorescein, was mainly in the stele. IAA exhibited a much higher lateral mobility from stele to cortex than did carboxyfluorescein. Based on the calculation of moles probe/kg fw, IAA is 4 times more concentrated in the stele than in the cortex while CF is 24 times higher in concentration in the stele than in the cortex. The structure of the node and the mesocotyl regions just below the node, regions of maximum growth, were examined and plasmodesmatal structure and frequency in these regions determined. The plasmodesmatal frequency, about 3 per micrometer2, between all cell types of the mesocotyl was found to be about 5-8 fold higher than that found for the root. Hypotheses of lateral auxin transport are discussed.

The mechanism by which an asymmetric distribution of plant growth hormone is attained.

Zea mays (sweet corn) seedlings attain an asymmetric distribution of the growth hormone indole-3-acetic acid (IAA) within 3 minutes following a gravity stimulus. Both free and esterified IAA (that is total IAA) accumulate to a greater extent in the lower half of the mesocotyl cortex of a horizontally placed seedling than in the upper half. Thus, changes in the ratio of free IAA to ester IAA cannot account for the asymmetric distribution. Our studies demonstrate there is no de novo synthesis of IAA in young seedlings. We conclude that asymmetric IAA distribution is attained by a gravity-induced, potential-regulated gating of the movement of IAA from kernel to shoot and from stele to cortex. As a working theory, which we call the Potential Gating Theory, we propose that perturbation of the plant's bioelectric field, induced by gravity, causes opening and closing of transport channels in the plasmodesmata connecting the vascular stele to the surrounding cortical tissues. This results in asymmetric growth hormone distribution which results in the asymmetric growth characteristics of the gravitropic response.

Ligand protons in a frozen solution of copper histidine relax via a T1e -driven three-spin mechanism.

Davies electron-nuclear double resonance spectra can exhibit strong asymmetries for long mixing times, short repetition times, and large thermal polarizations. These asymmetries can be used to determine nuclear relaxation rates in paramagnetic systems. Measurements of frozen solutions of copper(L-histidine)(2) reveal a strong field dependence of the relaxation rates of the protons in the histidine ligand, increasing from low (g( parallel)) to high (g( perpendicular)) field. It is shown that this can be attributed to a concentration-dependent T(1e)-driven relaxation process involving strongly mixed states of three spins: the histidine proton, the Cu(II) electron spin of the same complex, and another distant electron spin with a resonance frequency differing from the spectrometer frequency approximately by the proton Larmor frequency. The protons relax more efficiently in the g( perpendicular) region, since the number of distant electrons able to participate in this relaxation mechanism is higher than in the g( parallel) region. Analytical expressions for the associated nuclear polarization decay rate Tau(een) (-1) are developed and Monte Carlo simulations are carried out, reproducing both the field and the concentration dependences of the nuclear relaxation.