Effect of the Urtica dioica agglutinin on germination and cell wall formation of Phycomyces blakesleeanus Burgeff

The lectin from stinging nettle rhizomes, Urtica dioica agglutinin (UDA), did not affect the evolution of wet and dry weight, protein, nucleic acid, ATP, cAMP and glycerol content during early germination of Phycomyces blakesleeanus spores. However, earlier investigations established a strongly reduced mycelial growth of several phytopathogenic fungi by this small plant lectin. Total uptake and incorporation of radioactive precursors showed no differences between UDA or control hyphae, but UDA significantly altered the distribution patterns of [¹⁴C]-glucose incorporated into the walls of Phycomyces blakesleeanus (more label was recovered in the chitin fraction). Moreover, a small but significant stimulation of chitin synthase and a similar inhibition of chitin deacetylase was found in cell wall preparations. These observations could lead to a better understanding of plant-pathogen interrelationships and to a further elucidation of cell wall structure in fungi.Abbreviations GlcNAc N-Acetylglucosamine - PDB potato dextrose broth - PMM Phycomyces minimal medium - UDA Urtica dioica agglutinin - TEA tri-ethyl-amine - DAB 1,4-diaminobutanone     

Change in floral nectar components from fresh to senescent flowers ofCapparis spinosa (Capparidaceae), a nocturnally flowering Mediterranean shrub

We studied the nectar characteristics in relation to flower age of the summer flowering Mediterranean shrubCapparis spinosa in three localities in Southern Greece. Anthesis was nocturnal. Nectar volume, concentration, and sucrose/hexose ratio varied with site, year, and between individual plants; amino acid concentration varied only with site. The sucrose/hexose ratio decreased considerably with flower age, while the glucose/fructose ratio remained constant (ca. 1), implying that nectar sucrose broke down in the course of anthesis. Sugar breakdown increased with water content of nectar. Amino acid concentration was strongly age-dependent: It was low in fresh flowers, relatively high in middle-aged ones (except aspartic acid that was extremely increased), and very high in senescent ones. We attribute the amino acid changes to phenomena related to flower senescence in the dark.     

An exogalacturonase from Aspergillus aculeatus able to degrade xylogalacturonan

Summary A pectic polysaccharide from soy was degraded by a crude extracellular preparation of Aspergillus aculeatus. Besides monomeric sugars, an unknown oligosaccharide was produced, which was purified and identified as the dimer -Xyl _p -(1,3)-GalA _p . The enzyme responsible for the release of this dimer was purified and characterized as an exogalacturonase, which was not hindered by side-chains of xylose.     

Purification and characterization of fructan: fructan fructosyl transferase from chicory (Cichorium intybus L.) roots

Fructan: fructan fructosyl transferase (FFT, EC 2.4.1.100) was purified from chicory (Cichorium intybus L. var. foliosum cv. Flash) roots by a combination of ammonium sulfate precipitation, concanavalin A affinity chromatography, and anion- and cation-exchange chromatography. This protocol produced a 60-fold purification and a specific activity of 14.5 mol·(mg protein) ^–1·min^–1. The mass of the enzyme was 69 kDa as estimated by gel filtration. On sodium dodecyl sulfatepolyacrylamide gel electrophoresis and mass spectrometry, 52-kDa and 17-kDa fragments were found, suggesting that the enzyme was a heterodimer. Optimal activity was found between pH 5.5 and 6.5. The enzyme used 1-kestose, 1,1-nystose, oligofructan and commercial chicory root inulin (degree of polymerization 10) as donors and acceptors. Sucrose was the best acceptor but could not be used as a donor. However, at higher concentrations sucrose acted as a competitive inhibitor for donors of FFT. 1-Kestose was the most efficient and 1,1-nystose the least efficient donor. The purified enzyme exhibited -fructosidase activity, specially at higher temperatures and lower substrate concentrations. The synthesis of fructans from 1-kestose decreased at higher temperatures (5–50°C). Therefore enzyme assays were performed at 0°C. The same fructan oligosaccharides, with a distribution similar to that observed in vivo, were obtained upon incubation of the enzyme with sucrose and commercial chicory root inulin.Abbreviations Con A concanavalin A - DP degree of polymerization - FFT fructan: fructan fructosyl transferase - Fru fructose - Glc glucose - Kes 1-kestose - MALDI-TOF MS matrix-assisted laser desorption ionisation time of flight mass spectrometry - Nys 1,1-nystose - pI isoelectric point - SST sucrose: sucrose fructosyl transferase - Suc sucrose The authors would like to thank E. Nackaerts for valuable assistance. W. Van den Ende is also grateful to the National Fund for Scientific Research (NFSR Belgium) for giving a grant for research assistants. P. Verhaert is a research associate of the NFSR. This work was also supported by grant OT/91/18 from the Research Fund K.U. Leuven.     

Selection of peptides inhibiting a beta-lactamase-like activity.

A library of random peptide sequences was used to select peptides that inhibit an anti-idiotypic catalytic Ig, immunoglobulin (IgG) 9G4H9, with a beta-lactamase-like activity. This library displays cyclic heptapeptides on the surface of bacteriophages and represents a collection of up to 4.5 x 109 peptides. The first selection step aimed at enriching the library in species that bind to the whole Ig molecule. The second step was to discriminate peptides that bind to part of the molecule other than the active site. Selected peptides were then screened by surface plasmon resonance analysis. Those displaying measurable Kd values were assayed for their ability to inhibit the catalytic Ig.     

An Ex vivo Culture System to Study Thyroid Development

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are produced by differentiated thyrocytes, organized in closed spheres called follicles, while calcitonin is synthesized by C-cells, interspersed in between the follicles and a dense network of blood capillaries. Although adult thyroid architecture and functions have been extensively described and studied, the formation of the “angio-follicular” units, the distribution of C-cells in the parenchyma and the paracrine communications between epithelial and endothelial cells is far from being understood.This method describes the sequential steps of mouse embryonic thyroid anlagen dissection and its culture on semiporous filters or on microscopy plastic slides. Within a period of four days, this culture system faithfully recapitulates in vivo thyroid development. Indeed, (i) bilobation of the organ occurs (for e12.5 explants), (ii) thyrocytes precursors organize into follicles and polarize, (iii) thyrocytes and C-cells differentiate, and (iv) endothelial cells present in the microdissected tissue proliferate, migrate into the thyroid lobes, and closely associate with the epithelial cells, as they do in vivo.Thyroid tissues can be obtained from wild type, knockout or fluorescent transgenic embryos. Moreover, explants culture can be manipulated by addition of inhibitors, blocking antibodies, growth factors, or even cells or conditioned medium. Ex vivo development can be analyzed in real-time, or at any time of the culture by immunostaining and RT-qPCR.In conclusion, thyroid explant culture combined with downstream whole-mount or on sections imaging and gene expression profiling provides a powerful system for manipulating and studying morphogenetic and differentiation events of thyroid organogenesis.     

Synthesis and Biological Evaluation of a New N4-(N-Formyl Peptide)- 2′,3′-Dideoxy-3′-Thiacytidine as Anti-Hiv Prodrug

Abstract

The synthesis of a new analogue of 2′,3′-dideoxy-3′-thiacytidine 9 covalently linked to an N-formyl methionyl leucyl phenylalanine peptide is described. This new prodrug analogue has been tested on the one hand as activator of human polymorphonuclear leukocytes (an EC₅₀ value of 1.8 10^?5 M was determined from dose-response curve for superoxide production) and on the other hand as inhibitor of the syncitium formation caused by HIV-1 in MT4-cells (IC₅₀ = 8.0± 0.8 μM). In so far as this new prodrug possesses these two biological properties, it represents a useful “chemical-head” capable of targeting specific receptors located on leukocytes membranes.     

Sortilin,a novel APOE receptor implicated in Alzheimer disease

In the brain, apolipoprotein E (APOE) delivers cholesterol-rich lipoproteins to neurons to support synaptogenesis and maintenance of synaptic connections. Three APOE alleles exist in the human population with ε4 being an Alzheimer disease (AD) risk gene and ε2 being protective relative to the common ε3 variant. Many hypotheses have been advanced concerning allele-specific effects of APOE on neurodegeneration including effects on Aβ clearance, synaptic transmission, or neurotoxicity. Central to most proposed APOE functions is its interaction with receptors that mediate cellular uptake of this ligand. Several members of the LDL receptor gene family have been implicated as APOE receptors in the (patho)physiology of APOE in the brain, yet their specific modes of action in AD remain controversial. Recently, the pro-neurotrophin receptor sortilin has been identified as a novel APOE receptor in neurons. Ablation of sortilin expression in mice results in accumulation of APOE and Aβ in the brain. Moreover, primary neurons lacking sortilin exhibit significantly impaired uptake of APOE/Aβ complexes. Despite increased brain APOE levels, sortilin-deficient animals recapitulate anomalies in brain lipid homeostasis seen in APOE null mice, indicating functional deficiency in APOE uptake pathways. Taken together, these findings suggest a link between Aβ catabolism and pro-neurotrophin signaling converging on this receptor pathway.