Formation of methionine sulfoxide by peroxynitrite at position 1606 of von Willebrand factor inhibits its cleavage by ADAMTS-13: A new prothrombotic mechanism in diseases associated with oxidative stress

An enhanced formation of reactive oxygen species and peroxynitrite occurs in several clinical settings including diabetes, coronary artery disease, stroke, sepsis, and chronic inflammatory diseases. Peroxynitrite oxidizes methionine and tyrosine residues to methionine sulfoxide (MetSO) and 3-nitrotyrosine (NT), respectively. Notably, ADAMTS-13 cleaves von Willebrand factor (VWF) exclusively at the Tyr1605–Met1606 peptide bond in the A2 domain. We hypothesized that peroxynitrite could oxidize either or both of these amino acid residues, thus potentially affecting ADAMTS-13-mediated cleavage. We tested our hypothesis using synthetic peptide substrates based on: (1) VWF Asp1596–Ala1669 sequence (VWF74) and (2) VWF Asp1596–Ala1669 sequence containing nitrotyrosine (VWF74-NT) or methionine sulfoxide (VWF74-MetSO) at position 1605 or 1606, respectively. The peptides were treated with recombinant ADAMTS-13 and the cleavage products analyzed by RP-HPLC. VWF74 oxidized by peroxynitrite underwent a severe impairment of its hydrolysis. Likewise, VWF74-MetSO was minimally hydrolyzed, whereas VWF74-NT was hydrolyzed slightly more efficiently than VWF74. Oxidation by peroxynitrite of purified VWF multimers inhibited ADAMTS-13 hydrolysis, but did not alter their electrophoretic pattern nor their ability to induce platelet agglutination by ristocetin. Moreover, VWF purified from type 2 diabetic patients showed oxidative damage, as revealed by enhanced carbonyl, NT, and MetSO content and was partially resistant to ADAMTS-13 hydrolysis. In conclusion, peroxynitrite may contribute to prothrombotic effects, hindering the proteolytic processing by ADAMTS-13 of high-molecular-weight VWF multimers, which have the highest ability to bind and activate platelets in the microcirculation.     

Study protocol for a randomized controlled trial comparing mindfulness-based cognitive therapy with maintenance anti-depressant treatment in the prevention of depressive relapse/recurrence: the PREVENT trial

Background

The total effect of a medication is the sum of its drug effect, placebo effect (meaning response), and their possible interaction. Current interpretation of clinical trials' results assumes no interaction. Demonstrating such an interaction has been difficult due to lack of an appropriate study design.

Methods

180 adults were randomized to caffeine (300 mg) or placebo groups. Each group received the assigned intervention described by the investigators as caffeine or placebo, in a randomized crossover design. 4-hour-area-under-the-curve of energy, sleepiness, nausea (on 100 mm visual analog scales), and systolic blood pressure levels as well as caffeine pharmacokinetics (in 22 volunteers nested in the caffeine group) were determined. Caffeine drug, placebo, placebo-plus-interaction, and total effects were estimated by comparing outcomes after, receiving caffeine described as placebo to receiving placebo described as placebo, receiving placebo described as caffeine or placebo, receiving caffeine described as caffeine or placebo, and receiving caffeine described as caffeine to receiving placebo described as placebo, respectively.

Results

The placebo effect on area-under-the-curve of energy (mean difference) and sleepiness (geometric mean ratio) was larger than placebo-plus-interaction effect (16.6 [95% CI, 4.1 to 29.0] vs. 8.4 [-4.2 to 21.0] mm*hr and 0.58 [0.39 to 0.86] vs. 0.69 [0.49 to 0.97], respectively), similar in size to drug effect (20.8 [3.8 to 37.8] mm*hr and 0.49 [0.30 to 0.91], respectively), and its combination with the later was larger than total caffeine effect (29.5 [11.9 to 47.1] mm*hr and 0.37 [0.22 to 0.64]). Placebo-plus-interaction effect increased caffeine terminal half-life by 0.40 [0.12 to 0.68] hr (P = 0.007).

Conclusions

Drug and placebo effects of a medication may be less than additive, which influences the interpretation of clinical trials. The placebo effect may increase active drug terminal half-life, a novel mechanism of placebo action.

Trial Registration

ClinicalTrials.gov identification number - NCT00426010.     

Dynamic changes in cell surface molecules are very early events in the differentiation of mesophyll cells from Zinnia elegans into tracheary elements

The Zinnia mesophyll cell system consists of isolated leaf mesophyll cells in culture that can be induced, by auxin and cytokinin, to transdifferentiate semi-synchronously into tracheary elements (TEs). This system has been used to establish the precise time point at which the TE cell fate becomes determined, and then changes have been looked for in cell-wall composition and architecture that are associated with the establishment of competence, determination, and differentiation with the transition from primary to secondary cell wall formation. At very early stages in this time course, changes in the repertoire of proteins and polysaccharides both in the cell wall and secreted into the culture medium were found. Changes in the secretion of pectic polysaccharides, xyloglucans and arabinogalactan proteins (AGPs) have been detected using the monoclonal antibodies JIM 7, CCRC-M1 and JIM 13, that recognize these three classes of cell-wall molecule, respectively. Twenty-four hours before secondary thickenings are visible, an AGP is present in the primary walls of a subpopulation of cells, and is secreted into the culture medium. This molecule is present in the secondary thickenings of mature TEs but not in their surrounding primary walls. Methyl-esterified pectic polysaccharides are present in all cell walls and are secreted into the culture medium throughout the time course of differentiation, though at an increased rate in inductive medium. However, sugar and linkage analysis of culture media shows that a relatively unbranched rhamnogalacturonan is enriched in inductive medium around the time of determination and increases rapidly in concentration. The amount of fucosylated xyloglucan in cell walls increases during the time course, but appears in inductive medium 24 h earlier than in control medium and may have a subtly different structure. The fucose-containing epitope on the xyloglucan disappears abruptly and entirely from inductive medium 6 h before any secondary thickenings are visible in the cells. The disappearance of the epitope is correlated with secretion of several hydrolytic enzyme activities. In Zinnia leaves, the mesophyll cell walls contain neither the fucosylated xyloglucan nor the AGP, although methylesterified pectin is present. All three epitopes are expressed in the vascular bundles, and the AGP is specifically localized in the xylem cells. Fucosylated xyloglucan is also present in the epidermal tissue, and the AGP is present in guard cells. The dynamic behaviour of these specific cell-wall molecules is tightly correlated with differentiation events in vitro, and can be clearly distinguished from the production of new wall material found in expanding and elongating cells. The precise timing of the appearance and disappearance of these proteins and polysaccharides compared with the point of cell-fate determination provides us with a series of cell-surface markers for cell states at very early times in the transdifferentiation pathway.     

The modulation of calcium currents by the activation of mGluRs

Glutamatergic transmission in the central nervous system (CNS) is mediated by ionotropic, ligand-gated receptors (iGluRs), and metabotropic receptors (mGluRs). mGluRs are coupled to GTP-binding regulatory proteins (G-proteins) and modulate different second messenger pathways. Multiple effects have been described following their activation; among others, regulation of fast synaptic transmission, changes in synaptic plasticity, and modification of the threshold for seizure generation. Some of the major roles played by the activation of mGluRs might depend on the modulation of high-voltage-activated (HVA) calcium (Ca²⁺) currents. Some HVA Ca²⁺ channels (N-, P-, and Q-type channels) are signaling components at most presynaptic active zones. Their mGluR-mediated inhibition reduces synaptic transmission. The interference, by agonists at mGluRs, on L-type channels might affect the repetitive neuronal firing behavior and the integration of complex events at the somatic level. In addition, the mGluR-mediated effects on voltagegated Ca²⁺ signals have been suggested to strongly influence neurotoxicity. Rather different coupling mechanisms underlie the relation between mGluRs and Ca²⁺ currents: Together with a fast, membrane-delimited mechanism of action, much slower responses, involving intracellular second messengers, have also been postulated. In the recent past, the relative paucity of selective agonists and antagonists for the different subclasses of mGluRs had hampered the clear definition of the roles of mGluRs in brain function. However, the recent availability of new pharmacological tools is promising to provide a better understanding of the neuronal functions related to different mGluR subtypes. The analysis of the mGluR-mediated modulation of Ca²⁺ conductances will probably offer new insights into the characterization of synaptic transmission and the development of neuroprotective agents.     

Disease-associated N-terminal complement factor H mutations perturb cofactor and decay-accelerating activities

Many mutations associated with atypical hemolytic uremic syndrome (aHUS) lie within complement control protein modules 19-20 at the C terminus of the complement regulator factor H (FH). This region mediates preferential action of FH on self, as opposed to foreign, membranes and surfaces. Hence, speculation on disease mechanisms has focused on deficiencies in regulation of complement activation on glomerular capillary beds. Here, we investigate the consequences of aHUS-linked mutations (R53H and R78G) within the FH N-terminal complement control protein module that also carries the I62V variation linked to dense-deposit disease and age-related macular degeneration. This module contributes to a four-module C3b-binding site (FH1-4) needed for complement regulation and sufficient for fluid-phase regulatory activity. Recombinant FH1-4(V62) and FH1-4(I62) bind immobilized C3b with similar affinities (K(D) = 10-14 μM), whereas FH1-4(I62) is slightly more effective than FH1-4(V62) as cofactor for factor I-mediated cleavage of C3b. The mutant (R53H)FH1-4(V62) binds to C3b with comparable affinity (K(D) ～12 μM) yet has decreased cofactor activities both in fluid phase and on surface-bound C3b, and exhibits only weak decay-accelerating activity for C3 convertase (C3bBb). The other mutant, (R78G)FH1-4(V62), binds poorly to immobilized C3b (K(D) >35 μM) and is severely functionally compromised, having decreased cofactor and decay-accelerating activities. Our data support causal links between these mutations and disease; they demonstrate that mutations affecting the N-terminal activities of FH, not just those in the C terminus, can predispose to aHUS. These observations reinforce the notion that deficiency in any one of several FH functional properties can contribute to the pathogenesis of this disease.     

Knock-down of both eIF4E1 and eIF4E2 genes confers broad-spectrum resistance against potyviruses in tomato

Background

The eukaryotic translation initiation factor eIF4E plays a key role in plant-potyvirus interactions. eIF4E belongs to a small multigenic family and three genes, eIF4E1, eIF4E2 and eIF(iso)4E, have been identified in tomato. It has been demonstrated that eIF4E-mediated natural recessive resistances against potyviruses result from non-synonymous mutations in an eIF4E protein, which impair its direct interaction with the potyviral protein VPg. In tomato, the role of eIF4E proteins in potyvirus resistance is still unclear because natural or induced mutations in eIF4E1 confer only a narrow resistance spectrum against potyviruses. This contrasts with the broad spectrum resistance identified in the natural diversity of tomato. These results suggest that more than one eIF4E protein form is involved in the observed broad spectrum resistance.

Methodology/Principal Findings

To gain insight into the respective contribution of each eIF4E protein in tomato-potyvirus interactions, two tomato lines silenced for both eIF4E1 and eIF4E2 (RNAi-4E) and two lines silenced for eIF(iso)4E (RNAi-iso4E) were obtained and characterized. RNAi-4E lines are slightly impaired in their growth and fertility, whereas no obvious growth defects were observed in RNAi-iso4E lines. The F1 hybrid between RNAi-4E and RNAi-iso4E lines presented a pronounced semi-dwarf phenotype. Interestingly, the RNAi-4E lines silenced for both eIF4E1 and eIF4E2 showed broad spectrum resistance to potyviruses while the RNAi-iso4E lines were fully susceptible to potyviruses. Yeast two-hybrid interaction assays between the three eIF4E proteins and a set of viral VPgs identified two types of VPgs: those that interacted only with eIF4E1 and those that interacted with either eIF4E1 or with eIF4E2.

Conclusion/Significance

These experiments provide evidence for the involvement of both eIF4E1 and eIF4E2 in broad spectrum resistance of tomato against potyviruses and suggest a role for eIF4E2 in tomato-potyvirus interactions.     

Phenolic derivatives from Ruprechtia polystachya and their inhibitory activities on the glucose-6-phosphatase system

Two new compounds, 5-methyl-2-(2-methylbutanoyl)phloroglucinol 1-O-(6-O-β-D-apiofuranosyl)-β-D-glucopyranoside (1) and trans-2,3-dihydrokaempferol 3-O-(4-O-sulfo)-α-L-arabinopyranoside (2), together with 14 known flavonoids, trans-dihydrokaempferol 3-O-α-L-arabinopyranoside (3), trans-taxifolin 3-O-α-L-arabinofuranoside (4), quercetin 3-O-α-L-rhamnopyranoside (5), quercetin 3'-O-α-L-arabinofuranoside (6), catechin 3-O-α-L-rhamnopyranoside (7), trans-taxifolin 3-O-α-L-arabinopyranoside (8), cis-dihydrokaempferol 3-O-α-L-arabinopyranoside (9), catechin (10), myricetin 3-O-α-L-rhamnopyranoside (11), quercetin 3-O-α-L-arabinopyranoside (12), quercetin 3-O-α-L-arabinofuranoside (13), quercetin 3-O-(3″-galloyl)-α-L-rhamnopyranoside (14), quercetin 3-O-(2″-galloyl)-α-L-rhamnopyranoside (15), and epicatechin 3-O-gallate (16), were isolated from the leaves of Ruprechtia polystachya Griseb. (Polygonaceae). Their structures were established on the basis of extensive 1D- and 2D-NMR experiments as well as MS analyses. All compounds, except 1, showed inhibition of the enzyme glucose-6-phosphatase in intact microsomes.     

Mechanism of GABAB receptor-induced BDNF secretion and promotion of GABAA receptor membrane expression

Recent studies have shown that GABA(B) receptors play more than a classical inhibitory role and can function as an important synaptic maturation signal early in life. In a previous study, we reported that GABA(B) receptor activation triggers secretion of brain-derived neurotrophic factor (BDNF) and promotes the functional maturation of GABAergic synapses in the developing rat hippocampus. To identify the signalling pathway linking GABA(B) receptor activation to BDNF secretion in these cells, we have now used the phosphorylated form of the cAMP response element-binding protein as a biological sensor for endogenous BDNF release. In the present study, we show that GABA(B) receptor-induced secretion of BDNF relies on the activation of phospholipase C, followed by the formation of diacylglycerol, activation of protein kinase C, and the opening of L-type voltage-dependent Ca(2+) channels. We further show that once released by GABA(B) receptor activation, BDNF increases the membrane expression of β(2/3) -containing GABA(A) receptors in neuronal cultures. These results reveal a novel function of GABA(B) receptors in regulating the expression of GABA(A) receptor through BDNF-tropomyosin-related kinase B receptor dependent signalling pathway.