EM-fold: de novo atomic-detail protein structure determination from medium-resolution density maps

Electron density maps of membrane proteins or large macromolecular complexes are frequently only determined at medium resolution between 4?? and 10??, either by cryo-electron microscopy or X-ray crystallography. In these density maps, the general arrangement of secondary structure elements (SSEs) is revealed, whereas their directionality and connectivity remain elusive. We demonstrate that the topology of proteins with up to 250 amino acids can be determined from such density maps when combined with a computational protein folding protocol. Furthermore, we accurately reconstruct atomic detail in loop regions and amino acid side chains not visible in the experimental data. The EM-Fold algorithm assembles the SSEs de novo before atomic detail is added using Rosetta. In a benchmark of 27 proteins, the protocol consistently and reproducibly achieves models with root mean square deviation values <3??.     

Spatial patterns of plant diversity below-ground as revealed by DNA barcoding

Our understanding of the spatial organization of root diversity in plant communities and of the mechanisms of community assembly has been limited by our ability to identify plants based on root tissue, especially in diverse communities. Here, we test the effectiveness of the plastid gene rbcL, a core plant DNA barcoding marker, for investigating spatial patterns of root diversity, and relate observed patterns to above-ground community structure. We collected 3800 root fragments from four randomly positioned, 1-m-deep soil profiles (two vertical transects per plot), located in an old-field community in southern Ontario, Canada, and extracted and sequenced DNA from 1531 subsampled fragments. We identified species by comparing sequences with a DNA barcode reference library developed previously for the local flora. Nearly 85% of sampled root fragments were successfully sequenced and identified as belonging to 29 plant species or species groups. Root abundance and species richness varied in horizontal space and were negatively correlated with soil depth. The relative abundance of taxa below-ground was correlated with their frequency above-ground (r = 0.73, P = 0.0001), but several species detected in root tissue were not observed in above-ground quadrats. Multivariate analyses indicated that diversity was highly structured below-ground, and associated with depth, root morphology, soil chemistry and soil texture, whereas little structure was evident above-ground. Furthermore, analyses of species co-occurrence indicates strong species segregation overall but random co-occurrence among confamilials. Our results provide insights into the role of environmental filtering and competitive interactions in the organization of plant diversity below-ground, and also demonstrate the utility of barcoding for the identification of plant roots.     

Superoxide flux in endothelial cells via the chloride channel-3 mediates intracellular signaling

Reactive oxygen species (ROS) have been implicated in both cell signaling and pathology. A major source of ROS in endothelial cells is NADPH oxidase, which generates superoxide (O(2)(.-)) on the extracellular side of the plasma membrane but can result in intracellular signaling. To study possible transmembrane flux of O(2)(.-), pulmonary microvascular endothelial cells were preloaded with the O(2)(.-)-sensitive fluorophore hydroethidine (HE). Application of an extracellular bolus of O(2)(.-) resulted in rapid and concentration-dependent transient HE oxidation that was followed by a progressive and nonreversible increase in nuclear HE fluorescence. These fluorescence changes were inhibited by superoxide dismutase (SOD), the anion channel blocker DIDS, and selective silencing of the chloride channel-3 (ClC-3) by treatment with siRNA. Extracellular O(2)(.-) triggered Ca(2+) release in turn triggered mitochondrial membrane potential alterations that were followed by mitochondrial O(2)(.-) production and cellular apoptosis. These "signaling" effects of O(2)(.-) were prevented by DIDS treatment, by depletion of intracellular Ca(2+) stores with thapsigargin and by chelation of intracellular Ca(2+). This study demonstrates that O(2)(.-) flux across the endothelial cell plasma membrane occurs through ClC-3 channels and induces intracellular Ca(2+) release, which activates mitochondrial O(2)(.-) generation.     

Immunological Studies in Ugandan Patients with Hepatocellular Carcinoma

Immunological studies were performed on Ugandan patients with hepatocellular carcinoma to test the hypothesis that the high rate of persistence of hepatitis-associated antigen in these patients is the result of defects in host immune response. The responses to 1-chloro-2,4-dinitrobenzene sensitization and to a battery of recall skin test antigens were normal, as was the humoral antibody response to tularaemia antigen. Neither hypogammaglobulinaemia nor specific immunoglobulin deficiencies were found. Thus it appears unlikely that generalized defects in host immune responses can account for the high incidence of persistent hepatitis B virus infection found in Ugandan patients with hepatocellular carcinoma.     

Prostaglandins involvement in the formation of luteinized unrupted follicles in the cyclic female rat

The purpose of this investigation was to study the role played by prostaglandins in advanced ovulation and in the formation of luteinized unrupted follicles (LUF) in cyclic female rats. Dose related effects on ovulation were observed in rats given LH on diestrus 2 at 16.30. A significant positive correlation was observed between the number of postovulatory corpura lutea (POCL) and the increasing doses of LH. By contrast the number of LUF was negatively correlated with LH. Indomethacin treatment by 6h30 after administration of an ovulatory LH dose significantly increased the occurence of LUF at the expense of POCL. Conversely PGF_2α when admiststered by 6h30 after a subovulatory LH stimulation enhanced in a dose dependent manner the number of POCL with respect to the LH treated controls. Under a similar treatment with a subovulatory dose of LH, PGE₂ remained without ovulatory effects. The mechanisms of the formation of LUF are discussed on the basis of these results.     

Technical Factors Affecting an Immunoelectrophoretic Reference System for Analysis of Mycobacterial Antigens

The length of immunoelectrophoretic separation patterns obtained with a reference mycobacterial antigen was reduced significantly when a continuous buffer system was used in place of the recommended discontinuous system. Varying the supporting medium by using 1% concentrations of different agar preparations in the discontinuous system also affected the separation length and resolution of the reference pattern. Measurement of the electrical resistivity of the agar medium provided an explanation of these differences on the basis of current density or field intensity applied per slide. Variation in the reference pattern obtained in differently designed electrophoresis chambers also was attributed to differences in field intensity. To avoid these obvious alterations in the separation pattern of the reference antigen, it was suggested that separation procedures described originally for the reference system be used without modification.     

Modulation of chondrocyte proliferation by ascorbic acid and BMP-2

Chondrocytes show an unusual ability to thrive under serum-free conditions as long as insulin, thyroxine, and cysteine are present. Studies with sternal chondrocytes from chick embryos indicate that thymidine incorporation in chondrocytes cultured under serum-free conditions is 30–50% of that seen with fetal bovine serum (FBS). In contrast, skin fibroblast proliferation in serum-free culture is <5% of that seen with serum. Addition of 30–50 μM ascorbic acid to serum-free medium stimulates chondrocyte proliferation 4–5×, resulting in levels of thymidine incorporation higher than that seen with 10% serum. Three to five hours of ascorbate exposure is sufficient to stimulate proliferation, with maximal stimulation seen after 12–15 h. Bromo-deoxyuridine (BrdU) labelling indicated that approximately 25% of chondrocytes transit S phase during a 4-h period (16–20 h after ascorbate). Once maximal stimulation is reached, the proliferation rate remains fairly constant over at least 40 h. Ascorbate therefore increases the steady-state level of chondrocytes in the cycle. Because the stimulation of chondrocyte proliferation was greater than the net increase in cell numbers, we examined the level of apoptosis. Nuclear morphology, terminal uridine nucleotide end-labelling (TUNEL) assay, and 7-AAD/Hoechst dye FACS analyses all indicated that approximately 15% of the ascorbate-treated chondrocytes were undergoing apoptosis, while only 5% of the control chondrocytes were apoptotic. When prehypertrophic chondrocytes from the cephalic region of embryonic sternae were stimulated to undergo hypertrophy with rhBMP-2 + ascorbate, levels of apoptosis were similar to that seen with ascorbate alone. In contrast, treatment of caudal chondrocytes with BMP plus ascorbate does not induce hypertrophy, and the proportion of apoptotic cells was less than that seen with ascorbate alone. These results imply that in chondrocytes the transition to hypertrophy is associated with a decreased number of proliferating cells and a relatively high level of apoptosis. J. Cell. Physiol. 174:331–341, 1998. © 1998 Wiley-Liss, Inc.