Expression of syndecan, a putative low affinity fibroblast growth factor receptor, in the early mouse embryo.

Syndecan is an integral membrane proteoglycan that binds cells to several interstitial extracellular matrix components and binds to basic fibroblast-growth factor (bFGF) thus promoting bFGF association with its high-affinity receptor. We find that syndecan expression undergoes striking spatial and temporal changes during the period from the early cleavage through the late gastrula stages in the mouse embryo. Syndecan is detected initially at the 4-cell stage. Between the 4-cell and late morula stages, syndecan is present intracellularly and on the external surfaces of the blastomeres but is absent from regions of cell-cell contact. At the blastocyst stage, syndecan is first detected at cell-cell boundaries throughout the embryo and then, at the time of endoderm segregation, becomes restricted to the first site of matrix accumulation within the embryo, the interface between the primitive ectoderm and primitive endoderm. During gastrulation, syndecan is distributed uniformly on the basolateral cell surfaces of the embryonic ectoderm and definitive embryonic endoderm, but is expressed with an anteroposterior asymmetry on the surface of embryonic mesoderm cells, suggesting that it contributes to the process of mesoderm specification. In the extraembryonic region, syndecan is not detectable on most cells of the central core of the ectoplacental cone, but is strongly expressed by cells undergoing trophoblast giant cell differentiation and remains prominent on differentiated giant cells, suggesting a role in placental development. Immunoprecipitation studies indicate that the size of the syndecan core protein, although larger than that found in adult tissues (75 versus 69 x 10(3) Mr), does not change during peri-implantation development. The size distribution of the intact proteoglycan does change, however, indicating developmental alterations in its glycosaminoglycan composition. These results indicate potential roles for syndecan in epithelial organization of the embryonic ectoderm, in differential axial patterning of the embryonic mesoderm and in trophoblast giant cell function.     

Decoupling of the processes of molecular oxygen synthesis and electron transport in Ca2+-depleted PSII membranes

Extraction of Ca(2+) from the O(2)-evolving complex (OEC) of photosystem II (PSII) membranes with 2 M NaCl in the light (PSII(-Ca/NaCl)) results in 90% inhibition of the O(2)-evolution reaction. However, electron transfer from the donor to acceptor side of PSII, measured as the reduction of the exogenous acceptor 2,6-dichlorophenolindophenol (DCIP) under continuous light, is inhibited by only 30%. Thus, calcium extraction from the OEC inhibits the synthesis of molecular O(2) but not the oxidation of a substrate we term X, the source of electrons for DCIP reduction. The presence of electron transfer across PSII(-Ca/NaCl) membranes was demonstrated using fluorescence induction kinetics, a method that does not require an artificial acceptor. The calcium chelator, EGTA (5 mM), when added to PSII(-Ca/NaCl) membranes, does not affect the inhibition of O(2) evolution by NaCl but does inhibit DCIP reduction up to 92% (the reason why electron transport in Ca(2+)-depleted materials has not been noticed before). Another chelator, sodium citrate (citrate/low pH method of calcium extraction), also inhibits both O(2) evolution and DCIP reduction. The role of all buffer components (including bicarbonate and sucrose) as possible sources of electrons for PSII(-Ca/NaCl) membranes was investigated, but only the absence of chloride anions strongly inhibited the rate of DCIP reduction. Substitution of other anions for chloride indicates that Cl(-) serves its well-known role as an OEC cofactor, but it is not substrate X. Multiple turnover flash experiments have shown a period of four oscillations of the fluorescence yield (both the maximum level, F(max), and the fluorescence level measured 50 s after an actinic flash in the presence of DCMU) in native PSII membranes, reflecting the normal function of the OEC, but the absence of oscillations in PSII(-Ca/NaCl) samples. Thus, PSII(-Ca/NaCl) samples do not evolve O(2) but do transfer electrons from the donor to acceptor sides and exhibit a disrupted S-state cycle. We explain these results as follows. In Ca(2+)-depleted PSII membranes, obtained without chelators, the oxidation of the OEC stops after the absorption of three quanta of light (from the S1 state), which should convert the native OEC to the S4 state. An one-electron oxidation of the water molecule bound to the Mn cluster then occurs (the second substrate water molecule is absent due to the absence of calcium), and the OEC returns to the S3 state. The appearance of a sub-cycle within the S-state cycle between S3-like and S4-like states supplies electrons (substrate X is postulated to be OH(-)), explains the absence of O(2) production, and results in the absence of a period of four oscillation of the normal functional parameters, such as the fluorescence yield or the EPR signal from S2. Chloride anions probably keep the redox potential of the Mn cluster low enough for its oxidation by Y(Z)(*).     

The protective effect of lactose on lyophilization of CNK-20402

The goal of this research was to assess the feasibility of using lyophilization to stabilize an exploratory compound, CNK-20402, with a minimal amount of impurity (CNK-20193) formation. A mixed-level full factorial experimental design was used to screen excipients of glycine, mannitol, lactose monohydrate, and povidone K-12. Cryostage microscopy, powder X-ray diffraction, Karl Fischer titration, HPLC, and water vapor sorption were used to assess the formulations' physicochemical properties and stability. Initial physical characterization from powder X-ray diffraction revealed that the mannitol- and glycine-containing formulations were crystalline with the patterns of the pure excipient, whereas the remaining formulations were amorphous in structure. Chemically, the formulations stored at 50°C for 1 month had 2.36%, 1.05%, 0.81%, 0.79%, and 0.49% CNK-20193 for glycine, mannitol, drug alone, povidone K-12, and lactose formulations, respectively. The formulations containing drug-mannitol, drug alone, and druglactose were selected for accelerated stability study based on statistical analysis. Recovery of CNK-20193 in these formulations was 1.22%, 1.00%, and 0.55%, respectively, when stored at 40°C/75% relative humidity storage conditions for 3 months. Water vapor sorption analysis revealed weight gains of over 7%, 21%, and 24% for the mannitol, lactose, and drug alone formulations, respectively. Testing formulations with different concentrations of lactose by water vapor sorption indicated that CNK-20402 concentrations as low as 10% (wt/wt) could inhibit the recrystallization of lactose. The lactose-containing formulation exhibited the best stability among the formulations tested. The protective mechanism of lactose on the CNK-20402, based on water vapor sorption studies, is believed to be a result of (1) the drug-lactose interaction, and (2) competition between lactose and drug for the residual water in the formulation. Published: September 20, 2005     

Feedback equilibrium control during human standing

Equilibrium maintenance during standing in humans was investigated with a 3-joint (ankle, knee and hip) sagittal model of body movement. The experimental paradigm consisted of sudden perturbations of humans in quiet stance by backward displacements of the support platform. Data analysis was performed using eigenvectors of motion equation. The results supported three conclusions. First, independent feedback control of movements along eigenvectors (eigenmovements) can adequately describe human postural responses to stance perturbations. This conclusion is consistent with previous observations (Alexandrov et al., 2001b) that these same eigenmovements are also independently controlled in a feed-forward manner during voluntary upper-trunk bending. Second, independent feedback control of each eigenmovement is sufficient to provide its stability. Third, the feedback loop in each eigenmovement can be modeled as a linear visco-elastic spring with delay. Visco-elastic parameters and time-delay values result from the combined contribution of passive visco-elastic mechanisms and sensory systems of different modalities     

Finding gas diffusion pathways in proteins: application to O2 and H2 transport in CpI [FeFe]-hydrogenase and the role of packing defects

We report on a computational investigation of the passive transport of H2 and O2 between the external solution and the hydrogen-producing active site of CpI [FeFe]-hydrogenase from Clostridium pasteurianum. Two distinct methodologies for studying gas access are discussed and applied: (1) temperature-controlled locally enhanced sampling, and (2) volumetric solvent accessibility maps, providing consistent results. Both methodologies confirm the existence and function of a previously hypothesized pathway and reveal a second major pathway that had not been detected by previous analyses of CpI's static crystal structure. Our results suggest that small hydrophobic molecules, such as H2 and O2, diffusing inside CpI, take advantage of well-defined preexisting packing defects that are not always apparent from the protein's static structure, but that can be predicted from the protein's dynamical motion. Finally, we describe two contrasting modes of intraprotein transport for H2 and O2, which in our model are differentiated only by their size.     

Evidence that the plastid translocon Tic40 components possess modulating capabilities

The transport of proteins into the plastid is a process that faces changing cellular needs such as the situation found in different plant organs or developing tissues. The plastid translocon must therefore be responsive to the changing cell environment to deliver efficiently different arrays of structurally diverse proteins. Although the Tic40-related envelope proteins appear to be translocon components designed to address the varying needs of protein translocation, details of their involvement remain elusive. This study was thus designed to combine plant-based experiments and yeast mitochondrion-based approaches for unveiling clues related to how the Tic40 components may behave during the protein translocation process. The main findings related to how Tic40 proteins may work are: 1) natural fluctuations are apparent in developing tissues, in different organs of the same plant, and in different species; 2) transgenic Arabidopsis seedlings can tolerate functionally a wide range of variations in Tic40 levels, from partial suppression to excessive production; 3) the Tic40 proteins themselves exhibit configurational changes in their association with yeast mitochondria in response to different carbon sources; 4) the presence of Tic40 proteins in yeast mitochondria influences regulatory aspects of the mitochondrial translocon; and 5) the Tic40 proteins associate with mitochondrial translocon components involved in regulatory-like events. The combined data provide evidence that Tic40 proteins possess modulating capabilities.     

Reproducible polypeptide folding and structure prediction using molecular dynamics simulations

The folding of a polypeptide from an extended state to a well-defined conformation is studied using microsecond classical molecular dynamics (MD) simulations and replica exchange molecular dynamics (REMD) simulations in explicit solvent and in vacuo. It is shown that the solvated peptide folds many times in the REMD simulations but only a few times in the conventional simulations. From the folding events in the classical simulations we estimate an approximate folding time of 1-2 micros. The REMD simulations allow enough sampling to deduce a detailed Gibbs free energy landscape in three dimensions. The global minimum of the energy landscape corresponds to the native state of the peptide as determined previously by nuclear magnetic resonance (NMR) experiments. Starting from an extended state it takes about 50 ns before the native structure appears in the REMD simulations, about an order of magnitude faster than conventional MD. The calculated melting curve is in good qualitative agreement with experiment. In vacuo, the peptide collapses rapidly to a conformation that is substantially different from the native state in solvent.     

Role of Asn-16 and Ser-19 in anthopleurin B binding. Implications for the electrostatic nature of Na(V) site 3

Anthopleurin B (ApB) is a type 1 sea anemone toxin, which binds to voltage-sensitive sodium channels (Na(V)'s), thereby delaying channel inactivation. Previous work from our laboratories has demonstrated that the structurally unconstrained region involving residues 8-17 of this polypeptide, designated the Arg-14 loop, is important for full toxin affinity (Seibert et al., (2003) Biochemistry 42, 14515). Within this region, important contributions are made by residues Arg-12 and Leu-18 (Gallagher and Blumenthal, (1994) J. Biol. Chem. 269, 254; Dias-Kadambi et al., (1996) J. Biol. Chem. 271, 23828). Moreover, replacement of glycine residues found at positions 10 or 15 of the loop by alanine has been shown to have profound, isoform-selective effects on toxin-binding kinetics (Seibert et al., (2003)Biochemistry 42, 14515). To thoroughly understand the importance of this entire region, the work described here investigates the contribution of ApB residues Asn-16, Thr-17, and Ser-19 to toxin affinity and isoform selectivity. Our results demonstrate that residues within and proximal to the C terminus of the Arg-14 loop are important modulators of ApB affinity for Na(V) channels, indicating that the loop and channel site 3 are likely in close contact. A comparison of the effects of multiple replacements at each position reveals that Asn-16 and Ser-19 are involved in binding, whereas Thr-17 is not. The fact that anionic replacements for Asn-16 or Ser-19 are highly deleterious for toxin binding strongly suggests that site 3 contains either formal anionic residues or regions of high electron density, which could be formed by aromatic clusters. These data represent the first indication of the presence of such residues or regions within Na(V) site 3.     

Sampling and interpretation of psyllid nymph counts in potatoes

Development of effective management practices for insect pests relies heavily on sampling methods to accurately detect and quantify emerging populations. Herein we describe a novel method to extract and count nymphs of potato psyllid, Bactericera cockerelli (?ulc) (Hemiptera: Triozidae), from leaves in commercial fields of potato, Solanum tuberosum L. (Solanaceae). The proposed sampling method (referred to as the leaf washing method, LWM) consists of: (1) immersing samples of infested leaves in cold water to remove dust and sand; (2) immersing leaves in >85 °C water for 5 s; (3) extracting psyllid nymphs from heated water by passing it through a sampling unit composed of a funnel with a fine mesh‐organza fabric inside and attached to a vacuum pump; and (4) removing organza fabric and count psyllid nymphs under a stereoscope. With five sampling units operating simultaneously, one person is able to process and count immatures from about 400 leaf samples in two normal work days. As examples of applications, the LWM was used to characterize: (1) the vertical distribution of potato psyllid nymphs in the potato canopy; and (2) the spatio‐temporal distribution of potato psyllid nymphs in three potato varieties. Using LWM, we showed that psyllid nymphs were most predominant in the middle portion of the canopy and that spatio‐temporal distributions of nymphs varied among potato varieties.     

The Energy Return on Investment for Algal Biocrude: Results for a Research Production Facility

This study is an experimental determination of the energy return on investment (EROI) for algal biocrude production at a research facility at the University of Texas at Austin (UT). During the period of this assessment, algae were grown at several cultivation scales and processed using centrifugation for harvesting, electromechanical cell lysing, and a microporous hollow fiber membrane contactor for lipid separation. The separated algal lipids represent a biocrude product that could be refined into fuel and the post-extraction biomass could be converted to methane. To determine the EROI, a second-order analysis was conducted, which includes direct and indirect energy flows, but does not include energy expenses associated with capital investments. The EROI for the production process evaluated here was significantly less than 1, however, the majority of the energy consumption resulted from non-optimized growth conditions. While the experimental results do not represent an expected typical case EROI for algal fuels, the approach and end-to-end experimental determination of the different inputs and outputs provides a useful outline of the important parameters to consider in such an analysis. The Experimental Case results are the first known experimental energy balance for an integrated algal biocrude production facility, and as such, are expected to be helpful for setting research and development priorities. In addition to the Experimental Case (based on direct measurements), three analytical cases were considered in this work: (1) a Reduced (Inputs) Case, (2) a Highly Productive Case, and (3) a Literature Model. The Reduced (Inputs) Case and the Highly Productive Case speculate the energy use for a similar system in an improved, commercial-scale production setting. The Literature Model is populated with relevant data that have previously been reported in the literature. For the Experimental Case, Reduced Case, Highly Productive Case, and Literature Model, the estimated second-order EROI was 9.2?×?10^?4, 0.074, 0.22, and 0.35, respectively. These results were dominated by growth inputs (96%, 89%, 87%, and 61% of the total energy requirement, respectively). Furthermore, the EROI was adjusted using quality factors that were calculated according to the price of each input, yielding a quality-adjusted EROI that parallels a partial financial return on investment analysis. For the Experimental Case, the Reduced Case, and the Highly Productive Case, the quality-adjusted EROI was 9.2?×?10^?5, 0.013, and 0.36, respectively.