Sexual selection predicts brain structure in dragon lizards.

Phenotypic traits such as ornaments and armaments are generally shaped by sexual selection, which often favours larger and more elaborate males compared to females. But can sexual selection also influence the brain? Previous studies in vertebrates report contradictory results with no consistent pattern between variation in brain structure and the strength of sexual selection. We hypothesize that sexual selection will act in a consistent way on two vertebrate brain regions that directly regulate sexual behaviour: the medial preoptic nucleus (MPON) and the ventromedial hypothalamic nucleus (VMN). The MPON regulates male reproductive behaviour whereas the VMN regulates female reproductive behaviour and is also involved in male aggression. To test our hypothesis, we used high-resolution magnetic resonance imaging combined with traditional histology of brains in 14 dragon lizard species of the genus Ctenophorus that vary in the strength of precopulatory sexual selection. Males belonging to species that experience greater sexual selection had a larger MPON and a smaller VMN. Conversely, females did not show any patterns of variation in these brain regions. As the volumes of both these regions also correlated with brain volume (BV) in our models, we tested whether they show the same pattern of evolution in response to changes in BV and found that the do. Therefore, we show that the primary brain nuclei underlying reproductive behaviour in vertebrates can evolve in a mosaic fashion, differently between males and females, likely in response to sexual selection, and that these same regions are simultaneously evolving in concert in relation to overall brain size.

OS044. Morphological differences in murine placenta detected by magneticresonance imaging measurements of T2 relaxation times in mouse models ofpreeclampsia.

INTRODUCTION: We have demonstrated that morphologically distinct regions of the murine placenta can be detected by magnetic resonance imaging (MRI), with image contrast arising from the variation in T2 relaxation times between regions and dependent upon blood flow. Previous studies of human placenta by other groups have shown a homogeneous tissue with correlation of relaxation times with gestational age and a trend for shorter relaxation times in pregnancies complicated by preeclampsia and fetal growth restriction. The ability to detect morphological changes and alterations in blood flow in experimental models of preeclampsia would be a significant boost in understanding the relationship between abnormal placental implantation, reduced placental perfusion, inflammatory cytokines, angiogenic molecules and other factors that may play a role in the syndrome. OBJECTIVES: The aim of this study was to investigate whether morphological changes or abnormalities can be detected by T2 mapping in the placenta of mice subject to two experimental models of preeclampsia (reduced uterine perfusion pressure (RUPP) model and TNF-α induced model). METHODS: Pregnant C57BL/6JArc mice were, on day 13.5 of gestation, either subject to a unilateral ligation of the right uterine artery (RUPP) (n=2) or given an infusion of TNF-α by subcutaneous insertion of a mini-osmotic pump primed to deliver 500ng/kg/day for 4days (n=2). Controls were normal pregnant (n=2), sham-operated (n=1) or saline infused animals(n=1). MRI images were acquired on anaesthetised mice on day 17.5 of gestation using a Bruker Avance 11.7 Tesla wide-bore spectrometer with micro-imaging probe capable of generating gradients of 0.45T/m. T2 measurements were acquired using an MSME sequence protocol (Bruker MSME-T2-map) with an in-plane resolution of 0.1-0.2mm. Matlab was used to generate R2 (i.e.,1/T2) maps from the acquired data with the T2 values being calculated from selected regions of interest from 2-6 individual placenta from each mouse. RESULTS: Differences in the pattern of the regions of T2 contrast in the placenta were observed between normal, TNF-α treated and RUPP mice. The ratio of T2 values from the inner two regions was also significantly altered in TNF-α treated 1.98±0.09 (p=0.007); RUPP 1.94±0.11 (p=0.006) compared to normal animals 2.5±0.1 . CONCLUSION: These results demonstrate that morphological differences or abnormalities can be detected by T2 mapping in the placenta of mice subject to experimental models of preeclampsia and may be used to analyse changes quantitatively. This technology has the potential to be used when studying the dynamic changes in the placenta of pregnancies complicated by preeclampsia.

PP084. Magnetic resonance imaging measurements of T2 relaxation times within contrasting regions of murine placenta is dependent upon blood flow.

INTRODUCTION: It has been postulated that reduced placental perfusion as a result of abnormal placental implantation is the initiating event that leads to the maternal symptoms of preeclampsia. To be able to directly measure blood flow and perfusion in the placenta in experimental models of preeclampsia would provide valuable insight into the structural abnormalities of this syndrome. Magnetic resonance imaging (MRI) offers visualization of anatomy and analysis of changes in tissue morphology and function including blood flow and perfusion. The major source of image contrast in MRI comes from the variation in relaxation times between tissues. Previously, human placenta has appeared as fairly homogeneous in studies of T1 and T2 relaxation times, with no internal morphology apparent. OBJECTIVES: The aim of this study was to investigate, using much higher field strengths (11.7Tesla) and much higher resolution than have been used previously, whether structural inhomogeneities in the placenta can be discerned by T2 mapping and whether T2 mapping is capable of detecting structural abnormalities that may affect blood flow in a preeclamptic placenta. METHODS: Magnetic resonance images were acquired on an anaesthetised C57BL/6JArc mouse placed in a vertical animal probe using a Bruker Avance 11.7Tesla wide-bore spectrometer with micro-imaging probe capable of generating gradients of 0.45T/m. T2 measurements were acquired using an MSME sequence protocol (Bruker MSME-T2-map) with an in-plane resolution of 0.1-0.2mm. Matlab was used to generate R2 (i.e., 1/T2) maps from the acquired data with the T2 values being calculated from selected regions of interest within 5 individual placenta. Additional T2 measurements were acquired on the same slices immediately after blood flow was reduced to zero. RESULTS: Three distinct regions of T2 contrast were discerned in the mouse placenta, likely correlating to the labrynthine, junctional and decidual zones. The contrast between the inner two regions was substantially abrogated when blood flow ceased upon euthanasia of the animal by anaesthetic overdose (p<0.005), whereas the decidual region remained unchanged (p=0.13). CONCLUSION: These results demonstrate that morphologically distinct regions of the mouse placenta can be detected through the mapping of T2 relaxation times. The contrast between regions is lost when blood flow ceases and the placenta becomes homogeneous in appearance, suggesting that differences in T2 relaxation times across regions of the placenta may be used to non-invasively examine structural abnormalities and blood flow in the placenta of experimental animal models of preeclampsia.

Elastic and viscoelastic properties of porcine subdermal fat using MRI and inverse FEA.

There is a scarcity of investigation into the mechanical properties of subdermal fat. Recently, progress has been made in the determination of subdermal stress and strain distributions. This requires accurate constitutive modelling and consideration of the subdermal tissues. This paper reports the results of a study to estimate non-linear elastic and viscoelastic properties of porcine subdermal fat using a simple constitutive model. High-resolution magnetic resonance imaging (MRI) was used to acquire a time series of coincident images during a confined indentation experiment. Inverse finite element analysis was used to estimate the material parameters. The Neo Hookean model was used to represent the elastic behaviour (µ = 0.53 ± 0.31 kPa), while a single-element Prony series was used to model the viscoelastic response (α = 0.39 ± 0.03, τ = 700 ± 255 s).

Macroscopic background gradient and radiation damping effects on high-field PGSE NMR diffusion measurements.

The effects of macroscopic background gradients due to susceptibility differences at the sample interfaces and of radiation damping on pulsed-gradient spin-echo (PGSE) experiments are examined. Both phenomena can lead to the seemingly strange effect of the echo signal growing as the gradient strength increases at low applied gradient strengths. For a freely diffusing species, background gradients manifest themselves as slight concave or convex inflections in the linearized PGSE attenuation curve, depending on the polarity of the applied gradient. The various means of overcoming macroscopic background gradient problems, including bipolar gradients, and their efficacy are examined experimentally and discussed. The effects of radiation damping can also result in the attenuation curve being nonlinear but, different from the effect of background gradients, the nonlinearity does not change with the polarity of the applied gradient. The vulnerability of the stimulated echo-based PGSE sequence and variations of Hahn-based PGSE sequences is investigated. Both background gradients and radiation damping have serious implications for accurate diffusion measurement determination.

Time dependence of aggregation in crystallizing lysozyme solutions probed using NMR self-diffusion measurements.

The time dependence of aggregation in supersaturated lysozyme solutions was studied using pulsed-gradient spin-echo NMR diffusion measurements as a function of lysozyme concentration at pH 6.0 and 298 K in the presence of 0.5 M NaCl. The measurements provide estimates of the weight-averaged diffusion coefficient of the monomeric to intermediate molecular weight lysozyme species present in the solution (very large aggregates and crystals are excluded from the average due to the NMR relaxation-weighting effects inherent in the method). The results show that the average molecular weight of the various lysozyme aggregates changed with sigmoidal kinetics and that these kinetics were strongly influenced by the initial lysozyme concentration. The visualization of the time dependence of the protein aggregation afforded by this method provides a deeper understanding of how the crystallizing conditions (especially the initial protein concentration) are related to the resulting crystals.

Strategies for diagnosing and alleviating artifactual attenuation associated with large gradient pulses in PGSE NMR diffusion measurements.

The generation of phase-based artifacts resulting from mismatch in the effective areas (i.e., the time integrals) of sequential gradient pulses is discussed in the context of large gradient pulsed-gradient spin-echo (PGSE) NMR diffusion measurements. Such effects result in artifactual attenuation and distortion in the spectra which, in the first instance, are similar to (and commonly mistaken for) the effects of eddy currents. Small degrees of mismatch cause "unphysical" concave downward curvature in PGSE attenuation plots of freely diffusing species. However, larger mismatches can result in artifactual diffraction peaks in the plots which could easily be confused for true restricted diffusion effects. Although "rectangular" gradient pulses are preferable from a theoretical viewpoint, we found that shaped gradient (e.g., half-sine) pulses, which due to their slower rise and fall times were more tractable for the current amplifier, were more sequentially reproducible. As well as generating fewer phase-based artifacts such shaped pulses also decrease the likelihood of vibration problems.

Characterization of the solution properties of Pichia farinosa killer toxin using PGSE NMR diffusion measurements.

The solution behaviour with respect to pH and NaCl concentration of the tertiary structure and propensity for aggregation of salt- mediated killer toxin (SMKT) from Pichia farinosa was examined using pulsed-gradient spin-echo NMR diffusion measurements. It was found that in 0.15m NaCl the tertiary structure of SMKT was constant below pH 5.0, with the native SMKT existing as an unaggregated heterodimer containing the beta-subunit in a compactly folded form. However, above pH 5.0 the beta-subunit dissociated and lost its compact structure, becoming a random coil with an approximately 37% increase in effective hydrodynamic radius. To determine the effects of NaCl concentration on the tertiary structure of SMKT, diffusion measurements were performed at pH 3.5 and NaCl concentrations up to 2M. Both the tertiary structure and aggregation state of SMKT were found to be insensitive to the salt concentration which indicates that the activity of the toxin is not a direct result of salt-protein interactions.

A model for diffusive transport through a spherical interface probed by pulsed-field gradient NMR.

In biological systems, because of higher intracellular viscosity and/or the restriction of the diffusion space inside cells, the (apparent) diffusion coefficient of an intracellular species (e.g., water) is generally smaller than when it is in the extracellular medium. This difference affects the spin-echo signal attenuation in the pulsed field gradient NMR experiment and thus affords a means of separating the intracellular from the extracellular species, thereby providing a basis for studying transmembrane transport. Such experiments have commonly been analyzed using the macroscopic model of Kärger (see Adv. Magn. Reson. 21:1-89 (1988)). In our previous study, we considered a microscopic model of diffusive transport through a spherical interface using the short gradient pulse approximation (J. Magn. Reson. A114:39-46 (1995)). The spins in the external medium were modeled with the "partially absorbing wall" condition or as having a small but finite lifetime. In the present paper, we extend our treatment to the case in which there is no limitation upon the lifetime in either medium. We also consider a simple modification of Kärger's model that more properly accounts for the restricted intracellular diffusion. Importantly, it was found that the exact solution within the short gradient pulse approximation developed here and the modified Kärger model are in close agreement in the (experimentally relevant) long-time limit. The results of this study show that when there is no limitation upon the lifetime of the transported species in either phase, the spin-echo attenuation curve is very sensitive to transport.

Optimization of the water-PRESS pulse sequence and its integration into pulse sequences for studying biological macromolecules.

In this paper, the recently developed "Water-PRESS" method of water suppression [W. .S. Price and Y. Arata (1996), J. Magn. Reson. B 112, 190] in which homospoil pulses are used to manipulate the effects of radiation damping on the water resonance and thereby selectively alter the effective relaxation times of the water resonance with respect to the solute (e.g., biological macromolecules) resonances is further developed and applied. In the present work, methods for optimization in terms of degree of water suppression and in temporal terms (important for the application of Water-PRESS to multidimensional experiments) are considered so that recycle delays of less than 2.3 s (including the acquisition time) are possible. Also, a simple modification which allows the observation of solute resonances with relaxation times similar to that of the water resonance is presented. Finally, the inclusion into more complicated pulse sequences is also discussed. Experimental examples using aqueous samples of lysozyme and immunoglobulin are given. Compared to most other NMR water suppression techniques, this method is extremely simple to implement and optimize and does not require accurately calibrated RF pulses or perfect lineshape.

A pulsed field gradient NMR study of the aggregation and hydration of parvalbumin.

Pulsed field gradient NMR is a convenient alternative to traditional methods for measuring diffusion of biological macromolecules. In the present study, pulsed field gradient NMR was used to study the effects of calcium binding and hydration on carp parvalbumin. Carp parvalbumin is known to undergo large changes in tertiary structure with calcium loading. The diffusion coefficient is a sensitive guide to changes in molecular shape and in the present study the large changes in tertiary structure were clearly reflected in the measured diffusion coefficient upon calcium loading. The (monomeric) calcium-loaded form had a diffusion coefficient of 1.4 x 10(-10) m(2) s(-1) at 298 K, which conforms with the structure being a nearly spherical prolate ellipsoid from X-ray studies. The calcium-free form had a significantly lower diffusion coefficient of 1.1 x 10(-10) m(2) s(-1). The simplest explanation consistent with the change in diffusion coefficient is that the parvalbumin molecules form dimers upon the removal of Ca(2+) at the protein concentration studied (1 mM).

Visualization of Freezing Behaviors in Leaf and Flower Buds of Full-Moon Maple by Nuclear Magnetic Resonance Microscopy.

1H-Nuclear magnetic resonance (NMR) microscopy was used to study the freezing behavior of wintering buds of full-moon maple (Acer japonicum Thunb.). The images obtained predominantly reflected the density of mobile (i.e. non-ice) protons from unfrozen water. A comparison of NMR images taken at different subfreezing temperatures revealed which tissues produced high- and low-temperature exotherms in differential thermal analyses. In leaf and lower buds of A. japonicum, the scales and stem bark tissues were already frozen by -7[deg]C, but the primordial inflorescence and terminal primordial shoots remained supercooled at -14[deg]C, and the lateral primordial shoots were unfrozen even at -21[deg]C. The freezing of these supercooled tissues was associated with their loss of viability. The size of the supercooled primordial shoots and inflorescences was gradually reduced with decreasing temperature, indicating extraorgan freezing in these tissues. During this process the formation of dark regions beneath the primordia and subsequent gradual darkening in the basal part of supercooled primordia were visible. As the lateral shoot primordia were cooled, the unfrozen area was considerably reduced. Since the lateral primordia remained viable down to -40[deg]C, with no detectable low-temperature exotherms, they probably underwent type I extraorgan freezing. Deep supercooling in the xylem was clearly imaged. NMR microscopy is a powerful tool for noninvasively visualizing harmonized freezing behaviors in complex plant organs.

The effects of hypothermia on the intracellular pH of erythrocytes studied using 31P NMR and endogenous compounds.

The effects of hypothermia on the intracellular pH of human erythrocytes were studied non-invasively using 31P NMR spectroscopy and the endogenous phosphorus-containing compounds glycerate 2,3-bisphosphate and inorganic phosphate. Specifically, the pH dependence of the 31P NMR chemical shifts of these compounds was used to measure the intracellular pH at 25 and 37 degrees C. The possibility of a non-pH-dependent change on the chemical shifts of the 2-P and 3-P resonances of glycerate 2,3-bisphosphate due to the presence of paramagnetic deoxy-haemoglobin (i.e., a pseudo-contact interaction) was investigated and found to have negligible effect under the present experimental conditions. The most probable reasons for this are that the deoxy-haemoglobin concentration was too small and/or the glycerate 2,3-bisphosphate does not get sufficiently close to the paramagnetic centre to be affected. The change in intracellular pH with temperature was consistent with that predicted by the alphastat hypothesis.

Chloride-37 nuclear magnetic resonance spectroscopic study of binding of salicylic acid and other hydroxybenzoic acids to the band 3 anion transport protein of human erythrocytes.

Chloride-37 nuclear magnetic resonance spectroscopy was used to investigate the displacement of chloride (Cl-) from binding sites on band 3 anion transport protein in human erythrocytes by salicylic acid and five other hydroxybenzoic acids (HAs). All the HAs studied displaced Cl- from these binding sites. The association constants for binding of the HAs to band 3 anion transport protein were larger than that for Cl- and dependent on the specific structural features of the molecule, rather than general physicochemical characteristics.

Microviscosity of human erythrocytes studied using hypophosphite two-spin order relaxation.

A new 31P NMR method is used to probe the cytoplasmic viscosity of human erythrocytes. The method is based on observing two-spin order relaxation of the 31P atom of the hypophosphite ion. This method is superior to our previous method, using the longitudinal relaxation time of the ion, because random field effects such as intermolecular dipole-dipole relaxation can be separated from intramolecular relaxation. This allows a more accurate determination of the effective reorientational correlation time from the measured intramolecular relaxation because it is now unaffected by random field effects. The new method also provides a means by which to estimate the random field effects. Both two-spin order and proton-decoupled T1 measurements were conducted on hypophosphite in water solutions at various temperatures, glycerol solutions of various viscosities, and in erythrocyte samples of various cell volumes. The results show that the effective reorientational correlation time of the hypophosphite ion varies from 7.2 to 15.2 ps in the cytoplasm of cells ranging in volume from 102 to 56 fl cells.

A 35Cl and 37Cl NMR study of chloride binding to the erythrocyte anion transport protein.

Band 3, the erythrocyte anion transport protein, mediates the one-for-one exchange of bicarbonate and chloride ions across the membrane and consequently plays an important role in respiration. Binding to the protein forms the first step in the translocation of the chloride across the membrane. 35Cl and 37Cl NMR relaxation measurements at various field strengths were used to study chloride binding to the protein in the presence and absence of the transport inhibitor 4,4'-dinitrostilbene-2,2'-disulfonate. Significant differences occurred in the NMR relaxation rates depending on whether the inhibitor was present or not. The results indicate that the rate of chloride association and dissociation at each external binding site occurs on a time scale of less than or equal to 5 microseconds. This implies that the transmembrane flux is not limited by the rate of chloride binding to the external chloride binding site of band 3. The rotational correlation-time of chloride bound to band 3 was found to be greater than 20 ns with a quadrupole coupling constant of approximately 2 MHz.

Hypophosphite transport in human erythrocytes studied by overdetermined one-dimensional NMR exchange analysis.

The membrane transport kinetics of the disubstituted phosphorus oxyacid, hypophosphite, were studied in human red cells under equilibrium exchange conditions. Hypophosphite is an analogue of both the bicarbonate and phosphate ions and is known to be rapidly transported across the human red cell membrane via the anion transport protein, Band 3. The hypophosphite ion is a particularly useful probe of Band 3-mediated membrane transport as the intracellular and extracellular species occur as distinct resonances in the 31P NMR spectrum; as a result the membrane potential may also be readily inferred. We applied 'overdetermined' one-dimensional exchange analysis to estimate the rates of exchange for influx and efflux. The apparent equilibrium exchange (ee) values Kmee for the hypophosphite efflux and influx were different and while the efflux parameters were not able to be unambiguously defined, the measured apparent influx kinetic transport parameters were Vmaxee = 1600 +/- 190 amol cell-1 s-1, and Kmee = 75 +/- 16 mM.

Microviscosity of human erythrocytes studied with hypophosphite and 31P-NMR.

A 31P-NMR method, which complements earlier 13C-NMR procedures for probing the intra-erythrocyte microenvironment, is described. Hypophosphite is an almost unique probe of the erythrocyte microenvironment, since it is rapidly transported into the cell via the band 3 protein, and intra- and extracellular populations give rise to distinct resonances in the 31P-NMR spectrum. Relaxation mechanisms of the 31P nucleus in the hypophosphite ion were shown to be spin-rotation and dipole-dipole. Analysis of longitudinal relaxation rates in human erythrocytes, haemolysates and concentrated glycerol solutions allowed the determination of microviscosity using the Debye equation. Bulk viscosities of lysates and glycerol solutions were measured using Ostwald capillary viscometry. Translational diffusion coefficients were then calculated from the viscosity estimates using the Stokes-Einstein equation. The results with a range of solvent systems showed that 'viscosity' is a relative phenomenon and that bulk (i.e., macro-) viscosity is therefore not necessarily related to the NMR-determined viscosity. The intracellular NMR-determined viscosities from red cells, ranging in volume from 65.5 to 100.1 fl, varied from 2.10 to 2.67 mPa s. This is consistent with the translational diffusion coefficients of the hypophosphite ion altering by only 20%, whereas the values determined from bulk viscosity measurements conducted on lysates of these cells are consistent with a 230% change.