Survival of the external mycelium of a VAM fungus in frozen soil over winter

The present investigation examines (1) whether the external VAM mycelium survives winter freezing to act as a source of inoculum in the spring, and (2) whether soil disturbance reduces the infectivity of the external VAM mycelium following freezing of the soil. Sealed pouches of fine nylon mesh were placed in pots containing soil inoculated with a Glomus species. The mesh was impervious to roots but not to hyphae. Following two 3-week growth cycles of maize in the pots, the pouches were transplanted to the field. Pouches were removed from the field once during the 4 months when the soil was frozen, and once after spring thaw. Measurements were made of VAM spore density, hyphal length and viability in the pouches. Bioassays for infectivity were conducted on all pouches. Some VAM hyphae survived freezing and remained infective following winter freezing, in the absence of plant roots. Soil disturbance did not reduce the infectivity of hyphae following exposure to freezing temperatures. We observed a change in the distribution of viable cytoplasm within hyphae over winter, which we hypothesize represents an adaptation allowing hyphae to survive freezing temperatures. We suggest that the effect of disturbance on hyphal infectivity may be related to this seasonal change in the distribution of hyphal viability.     

Fluorescence labeling of the human erythrocyte anion transport system. Subunit structure studied with energy transfer

The anion transport system of human red cells was isolated in vesicles containing the original membrane lipids and the 95 000 dalton polypeptides (band 3) by the method of Wolosin et al. (J. Biol. Chem. (1977) 252, 2419–2427). The vesicles have a functional anion transport system since they display sulfate transport that is inhibited by the fluorescent probe 8-anilinonaphthalene 1-sulfonate (ANS) with similar potency as in red cells. The vesicles were labeled with the SH-specific probe fluorescein mercuric acetate (FMA). Labeling lowers FMA fluorescence, and is prevented or reversed by dithiothreitol, suggesting that the reaction is with a thiol group on the protein. Fluorescence titrations show a maximum labeling stoichiometry of 1.3 ± 0.4 mol FMA/mol 95 000 dalton polypeptide. The polarization of bound FMA fluorescence is high indicating that the probe is highly immobilized. Pretreatment with Cu²⁺ + o-phenanthroline under conditions that crosslink band 3 in ghosts decreases FMA labeling 50%. Differences in kinetics of FMA labeling in sealed and leaky vesicles suggest that the reactive SH group is located in the intravesicular portion of the protein (corresponding to the cytoplasmic surface of the red cell) and that FMA can cross the membrane. Inhibitors of anion transport have no effect on FMA labeling kinetics suggesting it is not transported via the anion     

Dissecting CNBP, a zinc-finger protein required for neural crest development, in its structural and functional domains

Cellular nucleic-acid-binding protein (CNBP) plays an essential role in forebrain and craniofacial development by controlling cell proliferation and survival to mediate neural crest expansion. CNBP binds to single-stranded nucleic acids and displays nucleic acid chaperone activity in vitro. The CNBP family shows a conserved modular organization of seven Zn knuckles and an arginine-glycine-glycine (RGG) box between the first and second Zn knuckles. The participation of these structural motifs in CNBP biochemical activities has still not been addressed. Here, we describe the generation of CNBP mutants that dissect the protein into regions with structurally and functionally distinct properties. Mutagenesis approaches were followed to generate: (i) an amino acid replacement that disrupted the fifth Zn knuckle; (ii) N-terminal deletions that removed the first Zn knuckle and the RGG box, or the RGG box alone; and (iii) a C-terminal deletion that eliminated the three last Zn knuckles. Mutant proteins were overexpressed in Escherichia coli, purified, and used to analyze their biochemical features in vitro, or overexpressed in Xenopus laevis embryos to study their function in vivo during neural crest cell development. We found that the Zn knuckles are required, but not individually essential, for CNBP biochemical activities, whereas the RGG box is essential for RNA-protein binding and nucleic acid chaperone activity. Removal of the RGG box allowed CNBP to preserve a weak single-stranded-DNA-binding capability. A mutant mimicking the natural N-terminal proteolytic CNBP form behaved as the RGG-deleted mutant. By gain-of-function and loss-of-function experiments in Xenopus embryos, we confirmed the participation of CNBP in neural crest development, and we demonstrated that the CNBP mutants lacking the N-terminal region or the RGG box alone may act as dominant negatives in vivo. Based on these data, we speculate about the existence of a specific proteolytic mechanism for the regulation of CNBP biochemical activities during neural crest development.     

Regulation of cell structure and function by actin-binding proteins: villin's perspective

Villin is a tissue-specific actin modifying protein that is associated with actin filaments in the microvilli and terminal web of epithelial cells. It belongs to a large family of actin-binding proteins which includes actin-capping, -nucleating and/or -severing proteins such as gelsolin, severin, fragmin, adseverin/scinderin and actin crosslinking proteins such as dematin and supervillin. Studies done in epithelial cell lines and villin knock-out mice have demonstrated the function of villin in regulating actin dynamics, cell morphology, epithelial-to-mesenchymal transition, cell migration and cell survival. In addition, the ligand-binding properties of villin (F-actin, G-actin, calcium, phospholipids and phospholipase C-gamma1) are mechanistically important for the crosstalk between signaling pathways and actin reorganization in epithelial cells.     

Mechanisms involved in the biosynthesis of polysaccharides

The mechanisms for the biosynthesis of three polysaccharides are presented: (i) starch synthesized by starch synthase and adenosine diphospho glucose; (ii) dextran synthesized by Leuconostoc mesenteroides B-512FMC dextransucrase and sucrose; and (iii) Acetobacter xylinum cellulose synthesized by cellulose synthase, uridine diphospho glucose, and bactoprenol phosphate. All three enzymes were pulsed with substrates, containing ¹⁴C-glucose and chased with the same nonlabeled substrates. When the polysaccharides were isolated, reduced, and hydrolyzed, the pulsed reactions gave ¹⁴C-glucitol, which was significantly decreased in the chase reaction. These experiments definitively show that all three polysaccharides are biosynthesized by the addition of glucose to the reducing-ends of the growing polysaccharides and not by the addition to the nonreducing-ends of primers. Additional evidence indicates that glucose and the polysaccharides are covalently attached to the active-sites of the enzymes. A two catalytic-site insertion mechanism at one active-site is proposed for the biosyntheses. Two of the polysaccharides are α-linked glucans, starch and dextran, and cellulose is a β-linked glucan, known for several years to require a bactoprenol lipid phosphate intermediate. It is shown how this intermediate is involved in determining that β-linkages are synthesized. Other β-linked polysaccharides: bacterial cell wall peptidomurein, Salmonella O-antigen polysaccharide, and Xanthanomonas camprestris xanthan, are heteropolysaccharides, with the later two also being hetero-linked polysaccharides, with the β-linkage at the reducing-end of the repeating unit. All three require bactoprenol lipid phosphate intermediates and are biosynthesized by the addition of the repeating units to the reducing-end of a growing polysaccharide chain, with the formation of a β-linkage.     

The pre-transmembrane region of the HCV E1 envelope glycoprotein: Interaction with model membranes

The previously identified membranotropic regions of the HCV E1 envelope glycoprotein, a class II membrane fusion protein, permitted us to identify different sequences which might be implicated in viral membrane fusion, membrane interaction and/or protein-protein binding. HCV E1 glycoprotein presents a membrano-active region immediately adjacent to the transmembrane segment, which could be involved in membrane destabilization similarly to the pre-transmembrane domains of class I fusion proteins. Consequently, we have carried out a study of the binding and interaction with the lipid bilayer of a peptide corresponding to segment 309-340, peptide E1_PTM, as well as the structural changes which take place in both the peptide and the phospholipid molecules induced by the binding of the peptide to the membrane. Here we demonstrate that peptide E1_PTM strongly partitions into phospholipid membranes, interacts with negatively-charged phospholipids and locates in a shallow position in the membrane. These data support its role in HCV-mediated membrane fusion and suggest that the mechanism of membrane fusion elicited by class I and II fusion proteins might be similar.     

Involvement of IL-17A in the pathogenesis of DSS-induced colitis in mice

To investigate the etiological implication of IL-17A in inflammatory bowel disease (IBD), dextran sodium sulfate (DSS) was administered to the mice deficient for the IL-17A gene. They showed only faint manifestations of colitis, as revealed by body weight loss, shrinkage in the colon length, serum haptoglobin concentration, and disease activity index. Although the mortality rate of WT mice reached approximately 60%, more than 90% of the IL-17A KO mice survived the DSS treatment. Histological change was also marginal in the IL-17A KO intestine, in which epithelial damage and inflammatory infiltrates were not obvious and the myeloperoxidase activity elevated only slightly. G-CSF and MCP-1 were abundantly produced in WT mouse intestine, whereas the production of these chemokines was drastically hampered in IL-17A-null intestine. The present results show that IL-17A plays a pivotal role in the pathogenesis of DSS-induced colitis, while MCP-1 and G-CSF may be crucially involved in the IL-17A-induced inflammation.     

Dual-channel photobleaching FRET microscopy for improved resolution of protein association states in living cells

Fluorescence resonance energy transfer (FRET) from a donor-labelled molecule to an acceptor-labelled molecule is a useful, proximity-based fluorescence tool to discriminate molecular states on the surface and in the interior of cells. Most microscope-based determinations of FRET yield only a single value, the interpretation of which is necessarily model-dependent. In this paper we demonstrate two new measurements of FRET heterogeneity using selective donor photobleaching in combination with synchronous donor/acceptor detection based on either (1) full kinetic analysis of donor-detected and acceptor-detected donor photobleaching or (2) a simple time-based ratiometric approach. We apply the new methods to study the cell surface distribution of concanavalin A yielding estimates of FRET and non-FRET population distributions, as well as FRET efficiencies within the FRET populations.     

Neural Connections Between Prosencephalic Structures Involved in Vasopressin Release

1. The diagonal band (DB) and the lateral septal area (LSA) are two prosencephalic structures, which were implicated in vasopressin release.2. The present experiment was designed to investigate neural connections between the DB and the LSA and from these nuclei to the paraventricular (PVN) and supraoptic (SON) nuclei, which could be related to vasopressin release.3. For the above purpose the bidirectional neuronal tracer biotinylated dextran amine (BDA) was injected into the DB or the LSA of male Wistar rats. Five days later the animals were sacrificed and brain slices were processed and analyzed to determine neuronal projections efferent from as well as afferent to these structures.4. Neuronal staining was more prominent in regions ipsilateral to the BDA injection site.5. After BDA injections into the DB, efferent projections from the DB were observed at the LSA, the PVN, the prefrontal cortex, the mediodorsal thalamic nucleus, and throughout the anterior hypothalamus, but not at the SON. At the PVN, labeled varicose fibers were observed at the magnocellular portion. The DB was found to receive a massive input from the LSA. More discrete projections to the DB were originated at the prefrontal cortex and from hypothalamic neurons outside the PVN and the SON.6. After BDA injections into the ventral portion of the LSA, efferent projections from the LSA were intense at the DB and throughout the hypothalamus. Labeled fibers were observed at the structures surrounding the SON or the PVN but not within those nuclei.7. The results indicate a massive neural output from the LSA to the DB and the existence of a direct neural connection from the DB to the PVN. No direct connections were observed between the LSA and the magnocellular nuclei (PVN and SON) or between the DB and the SON.     

Diffusion barriers of tripartite sporopollenin microcapsules prepared from pine pollen

Tripartite sporopollenin microcapsules prepared from pine pollen (Pinus sylvestris L. and Pinus nigra Arnold) were analysed with respect to the permeability of the different strata of the exine which surround the gametophyte and form the sacci. The sexine at the surface of the sacci is highly permeable for polymer molecules and latex particles with a diameter of up to 200 nm, whereas the nexine covering the gametophyte is impermeable for dextran molecules, with a Stokes' radius > or =4 nm (Dextran T 70), and for the tetravalent anionic dye Evans Blue (Stokes' radius = 1.3 nm). The central capsules obtained by dissolution of the sporoplasts showed strictly membrane-controlled exchange of non-electrolytes, with half-equilibration times in the range of minutes (monosaccharides, oligosaccharides) to hours (dextran molecules with Stokes' radii up to 2.5 nm). The dependence of the permeability coefficients of the nexine for non-electrolytes on Stokes' radius or molecular weight shows that the aqueous pores through the nexine are inhomogeneous with respect to their size, and that most pores are too narrow for free diffusion of sugar molecules. To explain the barrier function of the nexine for Evans Blue, it is assumed that at least the larger pores, which enable slow permeation of dextran molecules, contain negative charges.