Phi Values Research Articles

Ultraviolet Photolysis of HCHO: Absolute HCO Quantum Yields by Direct Detection of the HCO Radical Photoproduct

Absolute quantum yields for the radical (H + HCO) channel of HCHO photolysis, Phi(HCO), have been measured for the tropospherically relevant range of wavelengths (lambda) between 300 and 330 nm. The HCO photoproduct was directly detected by using a custom-built, combined ultra-violet (UV) absorption and cavity ring down (CRD) detection spectrometer. This instrument was previously employed for high-resolution (spectral resolution approximately 0.0035 nm) measurements of absorption cross-sections of HCHO, sigma(HCHO)(lambda), and relative HCO quantum yields. Absolute Phi(HCO) values were measured at seven wavelengths, lambda = 303.70, 305.13, 308.87, 314.31, 320.67, 325.59, and 329.51 nm, using an independent calibration technique based on the simultaneous UV photolysis of HCHO and Cl(2). These Phi(HCO) measurements display greater variability as a function of wavelength than the current NASA-JPL recommendations for Phi(HCO). The absolute Phi(HCO)(lambda) determinations and previously measured sigma(HCHO)(lambda) were used to scale an extensive set of relative HCO yield measurements. The outcome of this procedure is a full suite of data for the product of the absolute radical quantum yield and HCHO absorption cross-section, Phi(HCO)(lambda)sigma(HCHO)(lambda), at wavelengths from 302.6 to 331.0 nm with a wavelength resolution of 0.005 nm. This product of photochemical parameters is combined with high-resolution solar photon flux data to calculate the integrated photolysis rate of HCHO to the radical (H + HCO) channel, J(HCO). Comparison with the latest NASA-JPL recommendations, reported at 1 nm wavelength resolution, suggests an increased J(HCO) of 25% at 0 degrees solar zenith angle (SZA) increasing to 33% at high SZA (80 degrees). The differences in the calculated photolysis rate compared with the current HCHO data arise, in part, from the higher wavelength resolution of the current data set and highlight the importance of using high-resolution spectroscopic techniques to achieve a complete and accurate picture of HCHO photodissociation processes. All experimental Phi(HCO)(lambda)sigma(HCHO)(lambda) data are available for the wavelength range 302.6-331.0 nm (at 294 and 245 K and under 200 Torr of N(2) bath gas) as Supporting Information with wavelength resolutions of 0.005, 0.1, and 1.0 nm. Equivalent data sets of Phi(H(2)+CO)(lambda)sigma(HCHO)(lambda) for the molecular (H(2) + CO) photofragmentation channel, produced using the measured Phi(HCO)(lambda) sigma(HCHO)(tau) values, are also provided at 0.1 and 1.0 nm resolution.

Probing the free energy landscape of the FBP28WW domain using multiple techniques

The free-energy landscape of a small protein, the FBP 28 WW domain, has been explored using molecular dynamics (MD) simulations with alternative descriptions of the molecule. The molecular models used range from coarse-grained to all-atom with either an implicit or explicit treatment of the solvent. Sampling of conformation space was performed using both conventional and temperature-replica exchange MD simulations. Experimental chemical shifts and NOEs were used to validate the simulations, and experimental phi values both for validation and as restraints. This combination of different approaches has provided insight into the free energy landscape and barriers encountered by the protein during folding and enabled the characterization of native, denatured and transition states which are compatible with the available experimental data. All the molecular models used stabilize well defined native and denatured basins; however, the degree of agreement with the available experimental data varies. While the most detailed, explicit solvent model predicts the data reasonably accurately, it does not fold despite a simulation time 10 times that of the experimental folding time. The less detailed models performed poorly relative to the explicit solvent model: an implicit solvent model stabilizes a ground state which differs from the experimental native state, and a structure-based model underestimates the size of the barrier between the two states. The use of experimental phi values both as restraints, and to extract structures from unfolding simulations, result in conformations which, although not necessarily true transition states, appear to share the geometrical characteristics of transition state structures. In addition to characterizing the native, transition and denatured states of this particular system in this work, the advantages and limitations of using varying levels of representation are discussed.

Molecular dynamic simulations of nanomechanic chaperone peptide and effects of in silico His mutations on nanostructured function

The nanoscale peptide YSGVCHTDLHAWHGDWPLPVK exhibits molecular chaperone activity and prevents protein aggregation under chemical and/or thermal stress. Here, His mutations of this peptide and their impact on chaperone activity were evaluated using theoretical techniques. Molecular dynamic (MD) simulations with simulated annealing (SA) of different mutant nanopeptides were employed to determine the contribution of the scaffolding His residues (H45, H49, H52), when mutated to Pro, on chaperone action in vitro. The in silico mutations of His residues to Pro (H45P, H49P, H52P) revealed loss of secondary ordered strand structure. However, a small part of the strand conformation was formed in the middle region of the native chaperone peptide. The His-to-Pro mutations resulted in decreased gyration radius (Rg) values and surface accessibility of the mutant peptides under the simulation times. The invariant dihedral angle (phi) values and the disrupting effects of the Pro residues indicated the coil conformation of mutant peptides. The failure of the chaperone-like action in the Pro mutant peptides was consistent with their decreased effective accessible surfaces. The high variation of Phi value for His residues in native chaperone peptide leads to high flexibility, such as a minichaperone acting as a nanomachine at the molecular level. Our findings demonstrate that the peptide strand conformation motif with high flexibility at nanoscale is critical for chaperone activity.

Endemism and Regional Color and Genetic Differences in Five Putatively Cosmopolitan Reef Fishes

Endemism is thought to be relatively rare in marine systems due to the lack of allopatric barriers and the potential for long-distance colonization via pelagic larval dispersal. Although many species of coral reef fishes exhibit regionally restricted color variants that are suggestive of regional endemism, such variation is typically ascribed to intraspecific variation. We examined the genetic structure in 5 putatively monospecific fishes from the Indo-West Pacific (Amphiprion melanopus, Chrysiptera talboti, and Pomacentrus moluccensis [Pomacentridae] and Cirrhilabrus punctatus, and Labroides dimidiatus [Labridae]) that express regional color variation unique to this area. Mitochondrial-control-region sequence analysis showed shallow to deep genetic divergence in all 5 species (sequence divergence 2-17%), with clades concordant with regional color variation. These results were partially supported by nuclear RAG2 data. An analysis of molecular variation (AMOVA) mirrored the phylogenetic results; Phi(ST) values ranged from 0.91 to 0.7, indicating high levels of geographic partitioning of genetic variation. Concordance of genetics and phenotype demonstrate the genetic uniqueness of southwestern Pacific color variants, indicating that these populations are at a minimum distinct evolutionarily significant units and perhaps distinct regionally endemic species. Our results indicate that the alpha biodiversity of the southwestern Pacific is likely underestimated even in well-studied groups, such as reef fishes, and that regional endemism may be more common in tropical marine systems than previously thought.

Factors involved in alleviating water stress by partial crop removal in pear trees

We studied the relief of water stress associated with fruit thinning in pear (Pyrus communis L.) trees during drought to determine what mechanisms, other than stomatal adjustment, were involved. Combinations of control irrigation (equal to crop water use less effective rainfall) and deficit irrigation (equal to 20% of control irrigation), fruit load (unthinned and thinned to 40 fruits per tree) and root pruning (pruned and unpruned) treatments were applied to pear (cv. 'Conference') trees during Stage II of fruit development. Daily patterns of midday stem water potential (Psi(stem)) and leaf conductance to water vapor (g(l)) of deficit-irrigated trees differed after fruit thinning. In response to fruit thinning, gl progressively declined with water stress until 30 days after fruit thinning and then leveled off, whereas the effects of decreased fruit load on Psi(stem) peaked 30-40 days after fruit thinning and then tended to decline. Soil water depletion was significantly correlated with fruit load during drought. Our results indicate that stomatal adjustment and the resulting soil water conservation were the factors determining the Psi(stem) response to fruit thinning. However, these factors could not explain differences in daily patterns between g(l) and Psi(stem) after fruit thinning. In all cases, effects of root pruning treatments on Psi(stem) in deficit-irrigated trees were transitory (Psi(stem) recovered from root pruning in less than 30 days), but the recovery of Psi(stem) after root pruning was faster in trees with low fruit loads. This behavior is compatible with the concept that the water balance (reflected by Psi(stem) values) was better in trees with low fruit loads compared with unthinned trees, perhaps because more carbon was available for root growth. Thus, a root growth component is hypothesized as a mechanism to explain the bimodal Psi(stem) response to fruit thinning during drought.

IC-P2-142: Association of CSF total Tau and phospho-tau (181) with cerebral atrophy in mild cognitive impairment and Alzheimer's disease

Alzheimer’s disease is a hyperphosphorylated form of the microtubulebinding protein tau. Hyperphosphorylation of tau, which occurs significantly within the proline-rich domain, is associated with structural and functional changes that are poorly understood. Understanding the relationship between hyperphosphorylation of tau and structural changes in tau is a central question in the mechanism of neurofibrillary tangle formation. Methods: Peptides and larger protein constructs derived from the proline-rich region of tau as well as other tau domains were synthesized with defined phosphrylation states. The non-phosphorylated and phosphorylated peptides and proteins were analyzed by NMR and circular dichroism in order to identify the specific local changes in the structure of tau that are induced by hyperphosphorylation. Results: Phosphorylation of serine and threonine residues throughout the proline-rich domain of tau induced structural changes from a disordered conformation to a type II polyproline helix. The largest phosphorylation-induced structural changes were observed in tau peptides derived from residues 174-183 or residues 229-238. Phosphorylation-induced changes to polyproline helix were also observed in tau peptides incorporating residues 211-219. Phosphorylation of tau peptides derived from tau residues 196-209 resulted in the disruption of a nascent -helix. In these peptides, phosphoserine and phosphothreonine residues exhibited ordered values of phi ( ) (3J N 3.1-6.2 Hz; mean 4.7 Hz) compared to non-phosphorylated serine and threonine, indicating specific conformational restriction upon phosphorylation. Phosphorylation within the proline-rich domain did not affect the cis-trans isomerization equilibrium of the peptides examined, suggesting that phosphorylation-associated cis-trans isomerism is due to long-range interactions. As the proline-rich domains are linker regions between hydrophobic and aggregation-prone domains of tau, these results suggest that global reorganization of tau may occur upon hyperphosphorylation of proline-rich domain in tau. These results provide insight into possible mechanisms of phosphorylation-dependent structural changes of tau in Alzheimer’s disease. Conclusions: Hyperphosphorylation within the proline-rich domain of tau can induce specific local structural changes from a disordered conformation to an ordered polyproline II helix conformation.

Folding processes of the B domain of protein A to the native state observed in all-atom ab initio folding simulations

Reaching the native states of small proteins, a necessary step towards a comprehensive understanding of the folding mechanisms, has remained a tremendous challenge to ab initio protein folding simulations despite the extensive effort. In this work, the folding process of the B domain of protein A (BdpA) has been simulated by both conventional and replica exchange molecular dynamics using AMBER FF03 all-atom force field. Started from an extended chain, a total of 40 conventional (each to 1.0 micros) and two sets of replica exchange (each to 200.0 ns per replica) molecular dynamics simulations were performed with different generalized-Born solvation models and temperature control schemes. The improvements in both the force field and solvent model allowed successful simulations of the folding process to the native state as demonstrated by the 0.80 A C(alpha) root mean square deviation (RMSD) of the best folded structure. The most populated conformation was the native folded structure with a high population. This was a significant improvement over the 2.8 A C(alpha) RMSD of the best nativelike structures from previous ab initio folding studies on BdpA. To the best of our knowledge, our results demonstrate, for the first time, that ab initio simulations can reach the native state of BdpA. Consistent with experimental observations, including Phi-value analyses, formation of helix II/III hairpin was a crucial step that provides a template upon which helix I could form and the folding process could complete. Early formation of helix III was observed which is consistent with the experimental results of higher residual helical content of isolated helix III among the three helices. The calculated temperature-dependent profile and the melting temperature were in close agreement with the experimental results. The simulations further revealed that phenylalanine 31 may play critical to achieve the correct packing of the three helices which is consistent with the experimental observation. In addition to the mechanistic studies, an ab initio structure prediction was also conducted based on both the physical energy and a statistical potential. Based on the lowest physical energy, the predicted structure was 2.0 A C(alpha) RMSD away from the experimentally determined structure.

Microscopic Reversibility of Protein Folding in Molecular Dynamics Simulations of the Engrailed Homeodomain

The principle of microscopic reversibility states that at equilibrium the number of molecules entering a state by a given path must equal those exiting the state via the same path under identical conditions or, in structural terms, that the conformations along the two pathways are the same. There has been some indirect evidence indicating that protein folding is such a process, but there have been few conclusive findings. In this study, we performed molecular dynamics simulations of an ultrafast unfolding and folding protein at its melting temperature to observe, on an atom-by-atom basis, the pathways the protein followed as it unfolded and folded within a continuous trajectory. In a total of 0.67 micros of simulation in water, we found six transient denaturing events near the melting temperature (323 and 330 K) and an additional refolding event following a previously identified unfolding event at a high temperature (373 K). In each case, unfolding and refolding transition state ensembles were identified, and they agreed well with experiment on the basis of a comparison of S and Phi values. On the basis of several structural properties, these 13 transition state ensembles agreed very well with each other and with four previously identified transition states from high-temperature denaturing simulations. Thus, not only were the unfolding and refolding transition states part of the same ensemble, but in five of the seven cases, the pathway the protein took as it unfolded was nearly identical to the subsequent refolding pathway. These events provide compelling evidence that protein folding is a microscopically reversible process. In the other two cases, the folding and unfolding transition states were remarkably similar to each other but the paths deviated.

Riverine effects on mitochondrial structure of Bornean orang‐utans (Pongo pygmaeus) at two spatial scales

We examined mitochondrial DNA control region sequences of 73 Kinabatangan orangutans to test the hypothesis that the phylogeographical structure of the Bornean orangutan is influenced by riverine barriers. The Lower Kinabatangan Wildlife Sanctuary contains one of the most northern populations of orangutans (Pongo pygmaeus) on Borneo and is bisected by the Kinabatangan River, the longest river in Sabah. Orang-utan samples on either side of the river were strongly differentiated with a high Phi(ST) value of 0.404 (P < 0.001). Results also suggest an east-west gradient of genetic diversity and evidence for population expansion along the river, possibly reflecting a postglacial colonization of the Kinabatangan floodplain. We compared our data with previously published sequences of Bornean orangutans in the context of river catchment structure on the island and evaluated the general relevance of rivers as barriers to gene flow in this long-lived, solitary arboreal ape.

Reconstruction of the Sudden Oak Death epidemic in California through microsatellite analysis of the pathogen Phytophthora ramorum

The genetic structure of the clonally reproducing Sudden Oak Death (SOD) pathogen in California was investigated using seven variable microsatellites. A total of 35 multilocus genotypes were identified among 292 samples representative of populations from 14 forest sites and of the nursery trade. amova indicated significant genetic variability both within (44.34%) and among populations (55.66%). Spatial autocorrelation analyses indicated that Moran's index of similarity reached a minimum of 0.1 at 350 m, increased to 0.4 at 1500 m and then decreased to zero at 10 km. These results suggest a bimodal pattern of spread, with medium range dispersal (1500-10,000 m) putatively attributed to the presence of strong winds. Lack of genetic structure was identified for three groups of populations. One group notably included the nurseries' population and two forest populations, both linked to early reports of the pathogen. A neighbour-joining analysis based on pairwise Phi(ST) values indicated that the clade inclusive of the nurseries' populations is basal to all California populations. A network analysis identified three common genotypes as the likely founders of the California infestation and proposes a stepwise model for local evolution of novel genotypes. This was supported by the identification in the same locations of novel genotypes and of their 1- or 2-step parents. We hypothesize that the few undifferentiated population groups indicate historical human spread of the pathogen, while the general presence of genetically structured populations indicates that new infestations are currently generated by rare medium or long-range natural movement of the pathogen, followed by local generation of new genotypes.

Analysis of 3D structural differences in the IgG-binding domains based on the interresidue average-distance statistics

It is well-known that the IgG-binding domain from staphylococcal protein A folds into a 3 alpha helix bundle structure, while the IgG-binding domain of streptococcal protein G forms an (alpha + beta) structure. Recently, He et al. (Biochemistry 44:14055-14061, 2005) made mutants of these proteins from the wild types of protein A and protein G strains. These mutants are referred to as protein A219 and protein G311, and it was showed that these two mutants have different 3D structures, i.e., the 3 alpha helix bundle structure and the (alpha + beta) structure, respectively, despite the high sequence identity (59%). The purpose of our study was to clarify how such 3D structural differences are coded in the sequences with high homology. To address this problem, we introduce a predicted contact map constructed based on the interresidue average-distance statistics for prediction of folding properties of a protein. We refer to this map as an average distance map (ADM). Furthermore, the statistics of interresidue distances can be converted to an effective interresidue potential. We calculated the contact frequency of each residue of a protein in random conformations with this effective interresidue potential, and then we obtained values similar to phi values. We refer to this contact frequency of each residue as a p(mu) value. The comparison of the p(mu) values to the phi values for a protein suggests that p(mu) values reveal the information on the folding initiation site. Using these techniques, we try to extract the information on the difference in the 3D structures of protein A219 and protein G311 coded in their amino acid sequences in the present work. The results show that the ADM analyses and the p(mu) value analyses predict the information of folding initiation sites, which can be used to detect the 3D difference in both proteins.

Binding-Induced Folding of a Natively Unstructured Transcription Factor

Transcription factors are central components of the intracellular regulatory networks that control gene expression. An increasingly recognized phenomenon among human transcription factors is the formation of structure upon target binding. Here, we study the folding and binding of the pKID domain of CREB to the KIX domain of the co-activator CBP. Our simulations of a topology-based Gō-type model predict a coupled folding and binding mechanism, and the existence of partially bound intermediates. From transition-path and Φ-value analyses, we find that the binding transition state resembles the unstructured state in solution, implying that CREB becomes structured only after committing to binding. A change of structure following binding is reminiscent of an induced-fit mechanism and contrasts with models in which binding occurs to pre-structured conformations that exist in the unbound state at equilibrium. Interestingly, increasing the amount of structure in the unbound pKID reduces the rate of binding, suggesting a “fly-casting”-like process. We find that the inclusion of attractive non-native interactions results in the formation of non-specific encounter complexes that enhance the on-rate of binding, but do not significantly change the binding mechanism. Our study helps explain how being unstructured can confer an advantage in protein target recognition. The simulations are in general agreement with the results of a recently reported nuclear magnetic resonance study, and aid in the interpretation of the experimental binding kinetics.

Temperature‐dependent downhill unfolding of ubiquitin. I. Nanosecond‐to‐millisecond resolved nonlinear infrared spectroscopy

Transient thermal unfolding of ubiquitin is investigated using nonlinear infrared spectroscopy after a nanosecond laser temperature jump (T-jump). The abrupt change in the unfolding free energy surface and the ns time resolution allow us to observe a fast response on ns to micros time-scales, which we attribute to downhill unfolding, before a cross-over to ms kinetics. The downhill unfolding by a sub-population of folded proteins is induced through a shift of the barrier toward the native state. By adjusting the T-jump width, the effect of the initial (T(i)) and final (T(f)) temperature on the unfolding dynamics can be separated. From the amplitude of the fast downhill unfolding, the fractional population prepared at the unfolding transition state is obtained. This population increases with both T(i) and with T(f). A two-state kinetic analysis of the ms refolding provides thermodynamic information about the barrier height. By a combination of the fast and slow unfolding and folding parameters, a quasi-two-state kinetic analysis is performed to calculate the time-dependent population changes of the folded state. This calculation coincides with the experimentally obtained population changes at low temperature but deviations are found in the T-jump from 67 to 78 degrees C. Using temperature-dependent barrier height changes, a temperature Phi value analysis is performed. The result shows a decreasing trend of Phi(T) with temperature, which indicates an increase of the heterogeneity of the transition state. We conclude that ubiquitin unfolds along a well-defined pathway at low temperature which expands with increasing temperature to include multiple routes.

Extremely alkaline intracellular pH in the anterior stomach of larval yellow fever mosquitoes (Aedes aegypti)

The anterior stomach of larval Aedes aegypti is an alkalinizing organ where basolateral V‐ATPase drives luminal alkalinization to a pH of up to 12. However, the apical mechanisms involved are unknown. We hypothesized that luminal alkalinization involves absorption of H+ via apical H+ channels. Such a system would require increased intracellular pH (pHi) to function effectively. pHi was monitored using live cell imaging and BCECF as a pH‐dependent fluorophore. Serotonin (0.2 ìM), which stimulates luminal alkalinization, increased pHi in the anterior stomach from 7.0 ± 0.1 to 7.7 ± 0.1 (N = 64), whereas pHi was not affected in the posterior stomach (6.7 ± 0.1 before and 6.8 ± 0.1 after serotonin; N = 19). Perfusing the lumen with pH 10 saline increased pHi to 8.6 ± 0.1 (N = 41), suggesting a marked apical H+ permeability. However, in presence of luminal Zn2+ (10 ìM) similarly high pHi values were recorded (8.2 ± 0.1; N = 15), indicating that an apical proton channel, if present at all, cannot be blocked with Zn2+. The rate of pH change after switching to pH 10 in the lumen in the presence or absence of luminal Zn2+ was also not different. Together these results show that remarkably alkaline pHi values can be observed in the anterior stomach of larval Aedes aegypti. However, further investigation is required to determine the transporters involved in this unique physiological system. This work was supported by NIH (1R01AI063463‐01A2).

The influence of free time in the access to the practice of physical and sports activities in school age children

Introduction: With this study we aim to distinguish which are the free time variables associated to the adherence to physical and sports activities. Materials and Methods: A sample composed of 719 children aged between 6 and 10 years old, subject to the application of a questionnaire, was analyzed. The contingence tables and de Л (phi) value were used to verify the association between the free time variables and the practice of physical and sports activities. Results: The results' analysis allowed us to conclude that the places to spend free time (home, street, outdoor playground and public garden) and the time spent in these places are associated to the physical and sports activities. Discussion: There is a positive association between the time that children spend in outdoor playgrounds, street, public garden and the practice of global physical activities. In regards to the practice in clubs, we verifi ed a negative association between the time spent in the public garden, outdoor playground and street and the adherence to the practice. The time that they spend with video games is negatively associated to the formal practice. The time that they spend watching television is positively associated to the global practice, but negatively associated to the formal practice in business days.

Separation of a binary fluid mixture in a porous horizontal cavity

Thermogravitational separation has, until now, been used in differentially heated vertical cells, called thermogravitational columns (TGCs). The cell can be either a slit or an annular cavity whose two isothermal faces are maintained at different temperatures T1 and T2. In this study, we show contrary to what has been done until now, that it is possible to carry out the separation of the species of a binary mixture in the classical configuration of Rayleigh-Bénard (horizontal cell heated from below with the separation ratio psi>psi(mono)>0 or from above with psi<0); we obtain a monocellular flow for psi>psi(mono) at the onset of convection. The species separation, in a horizontal cell, is obtained without fear of the remixing observed in vertical thermogravitational cells for fluids with negative Soret coefficients. In this situation, the heaviest component concentrates in the upper part of the cell creating an unstable physical situation. We improve the efficiency of separation for the development of an industrial process of separation. We study the thermogravitational separation of the components of a binary fluid mixture saturating a horizontal porous layer, in the presence of the Soret effect. The horizontal walls of the cavity are impermeable and maintained at constant and different temperatures. The system of equations governing the problem has an equilibrium solution with a vertical stratification of temperature and concentration. This solution loses its stability via a stationary bifurcation for values of the separation ratio psi>psi(0) while, for psi<psi(0), stability is lost via a Hopf bifurcation. For a cavity heated from below, the flow resulting from the stationary bifurcation becomes monocellular beyond a certain value of psi>psi(mono)>0 and leads to a separation of the species between the two ends of the cell. We propose to determine whether the monocellular flow remains stable until the optimum of separation and if we can obtain high separation in the case of a horizontal cavity. We verify that the critical Rayleigh number Ra(c2), associated with the transition between monocellular flow and multicellular flow, is higher than the optimum Rayleigh number leading to maximum separation of species. Thus this study reveals that it is possible to obtain optimal separation before the monocellular flow loses its stability. We show also that the separation inside the horizontal cell in a Rayleigh-Bénard configuration permits us to produce the same degree of separation but with a greater quantity of each species compared to a thermogravitational vertical column.

The Folding Pathway of a Single Domain in a Multidomain Protein is not Affected by Its Neighbouring Domain

Domains are the structural, functional, and evolutionary components of proteins. Most folding studies to date have concentrated on the folding of single domains, but more than 70% of human proteins contain more than one domain, and interdomain interactions can affect both the stability and the folding kinetics. Whether the folding pathway is altered by interdomain interactions is not yet known. Here we investigated the effect of a folded neighbouring domain on the folding pathway of spectrin R16 (the 16th α-helical repeat from chicken brain α-spectrin) by using the two-domain construct R1516. The R16 folds faster and unfolds more slowly in the presence of its folded neighbour R15 (the 15th α-helical repeat from chicken brain α-spectrin). An extensive Φ-value analysis of the R16 domain in R1516 was completed to compare the transition state of the R16 domain alone with that of the R16 domain in a multidomain construct. The results indicate that the folding pathways are the same. This result validates the current approach of breaking up larger proteins into domains for the study of protein folding pathways.

Canine Anatomic Phantom for Preclinical Dosimetry in Internal Emitter Therapy

The majority of investigational studies of new diagnostic and therapeutic radiopharmaceuticals use murine animal models for preclinical assessments of pharmacokinetics and organ radiation dosimetry. Although mice and rats are widely available and relatively inexpensive, their smaller organ anatomy relative to that of humans can lead to considerable differences in organ dosimetry, thus complicating extrapolations of dose-response relationships to human patients. Nonhuman primates circumvent these problems in many respects but are increasingly becoming expensive and limited because of ethical considerations. With the recent completion of the dog genome project and the recognition of many similarities between canine and human cancers, dogs are increasingly being considered in cancer research and drug development. The main objective of this study was to construct a 3-dimensional computational phantom of a large dog on the basis of whole-body multislice CT data. A female hound cross underwent whole-body contrast-enhanced CT at a 2-mm slice thickness. On completion of the scan, the dog was euthanized, and the entire skeleton was harvested for a subsequent microCT investigation. The CT data were imported into a computational software program and used to create a polygon-mesh phantom of the entire animal. All of the major organs and bones were semiautomatically segmented and tagged to the CT slices. The phantom data were imported into a second software program and transformed to a nonuniform rational basis-spline surface phantom, allowing easy alteration of the phantom to simulate dogs of smaller or larger statures. A voxel-based version of the canine phantom was created by use of an in-house routine for subsequent import into the EGSnrc radiation transport code for photon and beta-particle organ dosimetry. The resulting voxel-based version of the canine phantom had a total body mass of 26.0 kg and a total body tissue mass (exclusive of wall organ content) of 24.5 kg. Although this University of Florida (UF) canine phantom displayed a total body mass intermediate between those of the Oak Ridge National Laboratory (ORNL) 5-y and 10-y stylized human phantoms of the MIRDOSE and OLINDA software codes, considerable differences were noted in organ photon cross-doses. For example, ratios of the specific absorbed fraction Phi(lungs <-- liver)(UF Dog) to Phi(lungs <-- liver)(ORNL 5-y) ranged from approximately 30 at 10 keV to approximately 3.5 at 1 MeV. Corresponding ratios of Phi(lungs <-- liver)(UF Dog) to Phi(lungs <-- liver)(ORNL 10-y) ranged from approximately 6 at 10 keV to approximately 1.3 at 1 MeV. Conversely, values of Phi(kidneys <-- spleen) and Phi(liver <-- spleen) were noted to be much lower (factors of 2-4) and much higher (factors of 2-15), respectively, in the canine phantom than in the ORNL human phantoms. These differences were attributed more to organ shape and position within the torso than to organ mass, because many of the canine organs closely approximated their counterparts volumetrically in the stylized pediatric human phantoms. The use of canine models, particularly in spontaneously occurring malignancies such as osteosarcoma, for preclinical testing of antineoplastic agents offers significant advantages over current murine models. However, the development of canine-specific technology is critical to the optimization of these studies. The UF canine dosimetry phantom described here aims to solve problems that could stem from the use of current human dosimetry models during radiopharmaceutical research.

Coordination of leaf and stem water transport properties in tropical forest trees

Stomatal regulation of transpiration constrains leaf water potential (Psi(L)) within species-specific ranges that presumably avoid excessive tension and embolism in the stem xylem upstream. However, the hydraulic resistance of leaves can be highly variable over short time scales, uncoupling tension in the xylem of leaves from that in the stems to which they are attached. We evaluated a suite of leaf and stem functional traits governing water relations in individuals of 11 lowland tropical forest tree species to determine the manner in which the traits were coordinated with stem xylem vulnerability to embolism. Stomatal regulation of Psi(L) was associated with minimum values of water potential in branches (Psi(br)) whose functional significance was similar across species. Minimum values of Psi(br) coincided with the bulk sapwood tissue osmotic potential at zero turgor derived from pressure-volume curves and with the transition from a linear to exponential increase in xylem embolism with increasing sapwood water deficits. Branch xylem pressure corresponding to 50% loss of hydraulic conductivity (P (50)) declined linearly with daily minimum Psi(br) in a manner that caused the difference between Psi(br) and P (50) to increase from 0.4 MPa in the species with the least negative Psi(br) to 1.2 MPa in the species with the most negative Psi(br). Both branch P (50) and minimum Psi(br) increased linearly with sapwood capacitance (C) such that the difference between Psi(br) and P (50), an estimate of the safety margin for avoiding runaway embolism, decreased with increasing sapwood C. The results implied a trade-off between maximizing water transport and minimizing the risk of xylem embolism, suggesting a prominent role for the buffering effect of C in preserving the integrity of xylem water transport. At the whole-tree level, discharge and recharge of internal C appeared to generate variations in apparent leaf-specific conductance to which stomata respond dynamically.

Purification, preliminary characterization and immunohistochemical localization of POSVP21 in the sand rat (Psammomys obesus) seminal vesicles

The sand rat Psammomys obesus is a mammalian species with male seasonal reproduction. Previously Gernigon et al. (1994) [Gernigon T, Berger M, Lecher P. Seasonal variations in the ultrastructure and production of androgen-dependent proteins in the seminal vesicles of a saharian rodent (Psammomys obesus). J Endocrinol 1994;142:37-46.] reported that the seminal vesicles of the adult sand rat contained a major secretory protein band (M.W. 21000) regulated by testosterone. This protein is synthesized in large amounts when the androgen level increases, and accounts for over 22% of soluble proteins from homogenate of seminal vesicles during the breeding season. When analyzed by NepHGE the protein band of 21kDa appeared to be composed of at least 3 visible spots with pHi values varying from 4 to 7. Its partially internal sequence was identified and exhibited five peptides. Polyclonal antibodies against POSVP21 were obtained in rabbits. They were also used to study immunohistochemical antigen localization by the means of an avidin-biotin peroxidase procedure. Observation showed that it is localized in the cytoplasm of epithelial cells and in secretory products in the lumen. The whole RNA of seminal vesicles was translated in a cell-free system derived from rabbit reticulocyte lysate and [35S]-methionine. Two major bands of 14.4 and 21kDa were visualized by means of denaturing gel electrophoresis. SDS-PAGE from medium incubation of seminal vesicle tissue with [35S]-methionine revealed one band with an apparent molecular weight of 21kDa. The results obtained indicate that seminal vesicle epithelium is the site of POSVP21 synthesis and the comparison of the partial amino acid composition of the internal sequence, indicated that POSVP21 constitute a family of most unusual proteins.