Beta-1,3 glucan sulfate, but not beta-1,3 glucan, induces the salicylic acid signaling pathway in tobacco and Arabidopsis

Sulfate substituents naturally occurring in biomolecules, such as oligosaccharides and polysaccharides, can play a critical role in major physiological functions in plants and animals. We show that laminarin, a beta-1,3 glucan with elicitor activity in tobacco (Nicotiana tabacum), becomes, after chemical sulfation, an inducer of the salicylic acid (SA) signaling pathway in tobacco and Arabidopsis thaliana. In tobacco cell suspensions, the oxidative burst induced by the laminarin sulfate PS3 was Ca2+ dependent but partially kinase independent, whereas laminarin triggered a strickly kinase-dependent oxidative burst. Cells treated with PS3 or laminarin remained fully responsive to a second application of laminarin or PS3, respectively, suggesting two distinct perception systems. In tobacco leaves, PS3, but not laminarin, caused electrolyte leakage and triggered scopoletin and SA accumulation. Expression of different families of Pathogenesis-Related (PR) proteins was analyzed in wild-type and mutant tobacco as well as in Arabidopsis. Laminarin induced expression of ethylene-dependent PR proteins, whereas PS3 triggered expression of ethylene- and SA-dependent PR proteins. In Arabidopsis, PS3-induced PR1 expression was also NPR1 (for nonexpressor of PR genes1) dependent. Structure-activity analysis revealed that (1) a minimum chain length is essential for biological activity of unsulfated as well as sulfated laminarin, (2) the sulfate residues are essential and cannot be replaced by other anionic groups, and (3) moderately sulfated beta-1,3 glucans are active. In tobacco, PS3 and curdlan sulfate induced immunity against Tobacco mosaic virus infection, whereas laminarin induced only a weak resistance. The results open new routes to work out new molecules suitable for crop protection.     

Kinetochore Localization of Spindle Checkpoint Proteins: Who Controls Whom?

The spindle checkpoint prevents anaphase onset until all the chromosomes have successfully attached to the spindle microtubules. The mechanisms by which unattached kinetochores trigger and transmit a primary signal are poorly understood, although it seems to be dependent at least in part, on the kinetochore localization of the different checkpoint components. By using protein immunodepletion and mRNA translation in Xenopus egg extracts, we have studied the hierarchic sequence and the interdependent network that governs protein recruitment at the kinetochore in the spindle checkpoint pathway. Our results show that the first regulatory step of this cascade is defined by Aurora B/INCENP complex. Aurora B/INCENP controls the activation of a second regulatory level by inducing at the kinetochore the localization of Mps1, Bub1, Bub3, and CENP-E. This localization, in turn, promotes the recruitment to the kinetochore of Mad1/Mad2, Cdc20, and the anaphase promoting complex (APC). Unlike Aurora B/INCENP, Mps1, Bub1, and CENP-E, the downstream checkpoint protein Mad1 does not regulate the kinetochore localization of either Cdc20 or APC. Similarly, Cdc20 and APC do not require each other to be localized at these chromosome structures. Thus, at the last step of the spindle checkpoint cascade, Mad1/Mad2, Cdc20, and APC are recruited at the kinetochores independently from each other.     

SNPs by AFLP (SBA): a rapid SNP isolation strategy for non-model organisms

Despite the great potential of single nucleotide polymorphism (SNP) markers in evolutionary studies, in particular for inferring population genetic parameters, SNP analysis has almost exclusively been limited to humans and ‘genomic model’ organisms, due to the lack of available sequence data in non-model organisms. Here, we describe a rapid and cost effective method to isolate candidate SNPs in non-model organisms. This SNP isolation strategy consists basically in the direct sequencing of amplified fragment length polymorphism bands. In a first application of this method, 10 unique DNA fragments that contained 24 SNPs were discovered in 11.11 kb of sequenced genomic DNA of a non-model species, the brown trout (Salmo trutta).     

Residues of VP26 of herpes simplex virus type 1 that are required for its interaction with capsids

VP26 is the smallest capsid protein and decorates the outer surface of the capsid shell of herpes simplex virus. It is located on the hexons at equimolar amounts with VP5. Its small size (112 amino acids) and high copy number make it an attractive molecule to use as a probe to investigate the complex pattern of capsid protein interactions. An in vitro capsid binding assay and a green fluorescent protein (GFP) localization assay were used to identify VP26 residues important for its interaction with capsids. To test for regions of VP26 that may be essential for binding to capsids, three small in-frame deletion mutations were generated in VP26, Delta18-25, Delta54-60, and Delta93-100. Their designations refer to the amino acids deleted by the mutation. The mutation at the C terminus of the molecule, which encompasses a region of highly conserved residues, abolished binding to the capsid and the localization of GFP to the nucleus in characteristic large puncta. Additional mutations revealed that a region of VP26 spanning from residue 50 to 112 was sufficient for the localization of the fused protein (VP26-GFP) to the nucleus and for it to bind to capsids. Using site-directed mutagenesis of conserved residues in VP26, two key residues for protein-protein interaction, F79 and G93, were identified as judged by the localization of GFP to nuclear puncta. When these mutations were analyzed in the capsid binding assay, they were also found to eliminate binding of VP26 to the capsid structure. Surprisingly, additional mutations that affected the ability of VP26 to bind to capsids in vitro were uncovered. Mutations at residues A58 and L64 resulted in a reduced ability of VP26 to bind to capsids. Mutation of the hydrophobic residues M78 and A80, which are adjacent to the hydrophobic residue F79, abolished VP26 capsid binding. In addition, the block of conserved amino acids in the carboxy end of the molecule had the most profound effect on the ability of VP26 to interact with capsids. Mutation of amino acid G93, L94, R95, R96, or T97 resulted in a greatly diminished ability of VP26 to bind capsids. Yet, all of these residues other than G93 were able to efficiently translocate or concentrate GFP into the nucleus, giving rise to the punctate fluorescence. Thus, the interaction of VP26 with the capsid appears to occur through at least two separate mechanisms. The initial interaction of VP26 and VP5 may occur in the cytoplasm or when VP5 is localized in the nucleus. Residues F79 and G93 are important for this bi-molecular interaction, resulting in the accumulation of VP26 in the nucleus in concentrated foci. Subsequent to this association, additional amino acids of VP26, including those in the C-terminal conserved domain, are important for interaction of VP26 with the three-dimensional capsid structure.     

Evidence of regio-specific glycosylation in human intestinal mucins: presence of an acidic gradient along the intestinal tract

Mucin glycans were isolated from different regions of the normal human intestine (ileum, cecum, transverse and sigmoid colon, and rectum) of two individuals with ALeb blood group. A systematic study of the monosaccharides and oligosaccharide alditols released by reductive beta-elimination from mucins was performed using gas chromatography, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, and nuclear magnetic resonance spectroscopy techniques. Important variations were observed in the mucin-associated oligosaccharide content with an increasing gradient of sialic acid from the ileum to the colon associated with a reverse gradient of fucose. Moreover, a comparative study of the Sda/Cad and ABH blood group determinants along the gastrointestinal tract showed the same reverse distribution in the two kinds of antigens. In addition, besides their heterogeneity, sialic acids presented considerable variations in the degree of O-acetylation in relation to glycan sialylation level. These data are discussed in view of recent concepts suggesting that the oligosaccharide composition of the gut constitutes a varied ecosystem for microorganisms that are susceptible to adapt there and possess the specific adhesion system and specific enzymes able to provide a carbohydrate nutrient.     

Evidence of a balance between phosphorylation and O-GlcNAc glycosylation of Tau proteins--a role in nuclear localization

Both phosphorylation and O-GlcNAc glycosylation posttranslationally modify microtubule-associated Tau proteins. Whereas the hyperphosphorylation of these proteins that occurs in Alzheimer's disease is well characterized, little is known about the O-GlcNAc glycosylation. The present study demonstrates that a balance exists between phosphorylation and O-GlcNAc glycosylation of Tau proteins, and furthermore that a dysfunction of this balance correlates with reduced nuclear localization.The affinity of Tau proteins for WGA lectin, together with evidence from [3H]-galactose transfer and analysis of beta-eliminated products, demonstrated the presence of O-GlcNAc residues on both cytosolic and nuclear Tau proteins. In addition, our data indicated the existence of a balance between phosphorylation and O-GlcNAc glycosylation events. Indeed, as demonstrated by 2D-electrophoresis and Western blotting, O-GlcNAc residues were mainly located on the less phosphorylated Tau 441 variants, whereas the more phosphorylated forms were devoid of O-GlcNAc residues. Furthermore, the Tau protein hyperphosphorylation induced by cellular okadaic acid treatment was correlated with reduced incorporation of O-GlcNAc residues into Tau proteins and with diminished Tau transfer into the nucleus. Hence, this paper establishes a direct relationship between O-GlcNAc glycosylation, phosphorylation and cellular localization of Tau proteins.     

Compressibility and uncoupling of cytochrome P450cam: high pressure FTIR and activity studies

The effect of the hydrostatic pressure on the CO ligand stretch vibration in cytochrome P450cam-CO bound with various substrates is studied by FTIR. The vibration frequency is linearily shifted to lower values with increasing pressure. The slope of the shift gives the isothermal compressibility of the heme pocket and is found to be related to the high-spin state content in an opposite direction to that previously observed from the pressure-induced shift of the Soret band. This opposite behaviour is explained by the dual effect of heme pocket water molecules both on the CO ligand and on electrostatic potentials produced by the protein at the distal side. The latter effect disturbs ligand-distal side contacts which are needed for a specific proton transfer in oxygen activation when dioxygen is the ligand. Their loss results in uncoupled H(2)O(2) formation.     

Cardiac autonomic control during balloon carotid angioplasty and stenting

Hemodynamic alterations during balloon carotid angioplasty (BCA) and stenting have been ascribed to the consequences of direct carotid baroreceptor stimulation during balloon inflation. BCA with stenting in patients with carotid atheromatous stenoses offers a unique opportunity for elucidating the cardiovascular autonomic response to direct transient intravascular stimulation of the baroreceptors. We analysed the consequences of BCA on the autonomic control of heart rate and on breathing components in nine patients with atheromatous stenoses involving the bifurcation and the internal carotid. A time-frequency domain method, the smoothed pseudo-Wigner-Ville transform (SPWVT), was used to evaluate the spectral parameters (i.e., the instantaneous amplitude and centre frequency (ICF) of the cardiovascular and respiratory oscillations). Those parameters and their dynamics (8 and 24 h later) were evaluated during and after the procedure. BCA stimulates baroreceptors in all patients, which markedly reduces heart rate and blood pressure. Vagal baroreflex activation altered the respiratory sinus arrhythmia in terms of amplitude and frequency (ICF HF RR shifted from 0.27 +/- 0.03 to 0.23 +/- 0.04 Hz pre-BCA vs. BCA, respectively; p < 0.01). Both the high- and low-frequency amplitudes of heart rate oscillations were altered during carotid baroreceptor stimulation, strongly supporting a contribution of the baroreflex to the generation of both oscillations of heart rate. Carotid baroreceptors stimulation increased the inspiratory time (Ti) (1.5 +/- 0.5 to 2.3 +/- 0.6 s pre-BCA vs. BCA, respectively; p < 0.01). In awake patients, BCA with stenting of atheromatous stenosis involving the bifurcation and internal carotid causes marked changes in the cardiac autonomic and respiratory control systems.