Role of the autonomic nervous system in generating non-linear dynamics in short-term heart period variability.

We evaluated the role played by the autonomic nervous system in producing non-linear dynamics in short heart period variability (HPV) series recorded in healthy young humans. Non-linear dynamics are detected using an index of predictability based on a local non-linear predictor and a surrogate data approach. Different types of surrogates are utilized: (i) phase-randomized Fourier-transform based (FT) data; (ii) amplitude-adjusted FT (AAFT) data; and (iii) iteratively refined AAFT (IAAFT) data of two types (IAAFT-1 and IAAFT-2). The approach was applied to experimental protocols activating or blocking the sympathetic or parasympathetic branches of the autonomic nervous system or periodically perturbing cardiovascular control via paced respiration at different breathing rates. We found that short-term HPV was mostly linear at rest. Experimental protocols activating the sympathetic or parasympathetic nervous system did not produce non-linear dynamics. In contrast, paced respiration, especially at slow breathing rates, elicited significantly non-linear dynamics. Therefore, in short-term HPV ( approximately 300 beats) the use of non-linear models is not supported by the data, except under conditions whereby the subject is constrained to a slow respiratory rate.     

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.     

The association of PNPLA3 variants with liver enzymes in childhood obesity is driven by the interaction with abdominal fat

Background and Aims

A polymorphism in adiponutrin/patatin-like phospholipase-3 gene (PNPLA3), rs738409 C->G, encoding for the I148M variant, is the strongest genetic determinant of liver fat and ALT levels in adulthood and childhood obesity. Aims of this study were i) to analyse in a large group of obese children the role of the interaction of not-genetic factors such as BMI, waist circumference (W/Hr) and insulin resistance (HOMA-IR) in exposing the association between the I148M polymorphism and ALT levels and ii) to stratify the individual risk of these children to have liver injury on the basis of this gene-environment interaction.

Methods

1048 Italian obese children were investigated. Anthropometric, clinical and metabolic data were collected and the PNPLA3 I148M variant genotyped.

Results

Children carrying the 148M allele showed higher ALT and AST levels (p = 0.000006 and p = 0.0002, respectively). Relationships between BMI-SDS, HOMA-IR and W/Hr with ALT were analysed in function of the different PNPLA3 genotypes. Children 148M homozygous showed a stronger correlation between ALT and W/Hr than those carrying the other genotypes (p: 0.0045) and, therefore, 148M homozygotes with high extent of abdominal fat (W/Hr above 0.62) had the highest OR (4.9, 95% C. I. 3.2–7.8, p = 0.00001) to develop pathologic ALT.

Conclusions

We have i) showed for the first time that the magnitude of the association of PNPLA3 with liver enzymes is driven by the size of abdominal fat and ii) stratified the individual risk to develop liver damage on the basis of the interaction between the PNPLA3 genotype and abdominal fat.     

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.     

d(CGGTGGT) forms an octameric parallel G-quadruplex via stacking of unusual G(:C):G(:C):G(:C):G(:C) octads

Among non-canonical DNA secondary structures, G-quadruplexes are currently widely studied because of their probable involvement in many pivotal biological roles, and for their potential use in nanotechnology. The overall quadruplex scaffold can exhibit several morphologies through intramolecular or intermolecular organization of G-rich oligodeoxyribonucleic acid strands. In particular, several G-rich strands can form higher order assemblies by multimerization between several G-quadruplex units. Here, we report on the identification of a novel dimerization pathway. Our Nuclear magnetic resonance, circular dichroism, UV, gel electrophoresis and mass spectrometry studies on the DNA sequence dCGGTGGT demonstrate that this sequence forms an octamer when annealed in presence of K(+) or NH(4)(+) ions, through the 5'-5' stacking of two tetramolecular G-quadruplex subunits via unusual G(:C):G(:C):G(:C):G(:C) octads.     

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.     

Circular dichroism studies of myoglobin and leghemoglobin.

The circular dichroism spectra of leghemoglobin a from the root nodules of soybean have been compared with those for sperm whale myoglobin in the fat- and near-ultraviolet and the Soret and visible regions of the spectrum. Circular dichroism spectra in the far-ultraviolet show that the leghemoglobins all have a high alpha-helix content (soybean leghemoglobin a, 55%) regardless of the nature of bound ligands and oxidation or spin state of the heme iron. The known sequence homologies with mammalian hemoglobins may therefore be reflected in conformational homologies as suggested by the x-ray studies of Vainshtein et al. ((1975) Nature (London) 254, 163-164) on lupin leghemoglobin. Removal of the heme moiety decreases helicity by only 9% for leghemoglobins, compared with 23% for myoglobin. This, the much smaller heme contribution to the near-ultraviolet circular dichroism than in myoglobin, and the greater accessibility of the heme moiety to aqueous solvent (Nicola et al. (1974), Proc. Aust. Biochem. Soc. 7, 21) suggest that the association between heme and protein is much weaker in leghemoglobins than in myoglobin. The aromatic Soret and visible circular dichroism spectra for all derivatives of leghemoglobin are opposite in sense to those for myoglobin, showing that the patterns of protein side chain contacts with the heme are different in the two classes of heme proteins. There is strong evidence that one of the two tryptophans whose identity and structural role in myoglobin is known, is present also in plant leghemoglobins, hydrogen-bonded and in a similar nonpolar environment whether heme is present or not. The above findings help to explain the remarkably high oxygen affinity and some other ligand-binding properties of leghemoglobins which differ from those of myoglobin.