Amino acid coevolution reveals three-dimensional structure and functional domains of insect odorant receptors.

Insect odorant receptors (ORs) comprise an enormous protein family that translates environmental chemical signals into neuronal electrical activity. These heptahelical receptors are proposed to function as ligand-gated ion channels and/or to act metabotropically as G protein-coupled receptors (GPCRs). Resolving their signalling mechanism has been hampered by the lack of tertiary structural information and primary sequence similarity to other proteins. We use amino acid evolutionary covariation across these ORs to define restraints on structural proximity of residue pairs, which permit de novo generation of three-dimensional models. The validity of our analysis is supported by the location of functionally important residues in highly constrained regions of the protein. Importantly, insect OR models exhibit a distinct transmembrane domain packing arrangement to that of canonical GPCRs, establishing the structural unrelatedness of these receptor families. The evolutionary couplings and models predict odour binding and ion conduction domains, and provide a template for rationale structure-activity dissection.

Differential contribution of mitochondria, NADPH oxidases, and glycolysis to region-specific oxidant stress in the anoxic-reoxygenated embryonic heart.

The ability of the developing myocardium to tolerate oxidative stress during early gestation is an important issue with regard to possible detrimental consequences for the fetus. In the embryonic heart, antioxidant defences are low, whereas glycolytic flux is high. The pro- and antioxidant mechanisms and their dependency on glucose metabolism remain to be explored. Isolated hearts of 4-day-old chick embryos were exposed to normoxia (30 min), anoxia (30 min), and hyperoxic reoxygenation (60 min). The time course of ROS production in the whole heart and in the atria, ventricle, and outflow tract was established using lucigenin-enhanced chemiluminescence. Cardiac rhythm, conduction, and arrhythmias were determined. The activity of superoxide dismutase, catalase, gutathione reductase, and glutathione peroxidase as well as the content of reduced and oxidized glutathione were measured. The relative contribution of the ROS-generating systems was assessed by inhibition of mitochondrial complexes I and III (rotenone and myxothiazol), NADPH oxidases (diphenylene iodonium and apocynine), and nitric oxide synthases (N-monomethyl-l-arginine and N-iminoethyl-l-ornithine). The effects of glycolysis inhibition (iodoacetate), glucose deprivation, glycogen depletion, and lactate accumulation were also investigated. In untreated hearts, ROS production peaked at 10.8 ± 3.3, 9 ± 0.8, and 4.8 ± 0.4 min (means ± SD; n = 4) of reoxygenation in the atria, ventricle, and outflow tract, respectively, and was associated with arrhythmias. Functional recovery was complete after 30-40 min. At reoxygenation, 1) the respiratory chain and NADPH oxidases were the main sources of ROS in the atria and outflow tract, respectively; 2) glucose deprivation decreased, whereas glycogen depletion increased, oxidative stress; 3) lactate worsened oxidant stress via NADPH oxidase activation; 4) glycolysis blockade enhanced ROS production; 5) no nitrosative stress was detectable; and 6) the glutathione redox cycle appeared to be a major antioxidant system. Thus, the glycolytic pathway plays a predominant role in reoxygenation-induced oxidative stress during early cardiogenesis. The relative contribution of mitochondria and extramitochondrial systems to ROS generation varies from one region to another and throughout reoxygenation.

The L-Type Ca+ and KATP channels may contribute to pacing-induced protection against anoxia-reoxygenation in the embryonic heart model.

L-Type Ca(2+) and K(ATP) Channels in Pacing-Induced Cardioprotection. AIMS: The L-type Ca(2+) channel, the sarcolemmal (sarcK(ATP)), and mitochondrial K(ATP) (mitoK(ATP)) channels are involved in myocardial preconditioning. We aimed at determining to what extent these channels can also participate in pacing-induced cardioprotection. METHODS: Hearts of 4-day-old chick embryos were paced in ovo during 12 hour using asynchronous intermittent ventricular stimulation at 110% of the intrinsic rate. Sham operated and paced hearts were then submitted in vitro to anoxia (30 minutes) and reoxygenation (60 minutes). These hearts were exposed to L-type Ca(2+) channel agonist Bay-K-8644 (BAY-K) or blocker verapamil, nonselective K(ATP) channel antagonist glibenclamide (GLIB), mitoK(ATP) channel agonist diazoxide (DIAZO), or antagonist 5-hydroxydecanoate. Electrocardiogram, electromechanical delay (EMD) reflecting excitation-contraction (E-C) coupling, and contractility were determined. RESULTS: Under normoxia, heart rate, QT duration, conduction, EMD, and ventricular shortening were similar in sham and paced hearts. During reoxygenation, arrhythmias ceased earlier and ventricular EMD recovered faster in paced hearts than in sham hearts. In sham hearts, BAY-K (but not verapamil), DIAZO (but not 5-hydroxydecanoate) or GLIB accelerated recovery of ventricular EMD, reproducing the pacing-induced protection. By contrast, none of these agents further ameliorated recovery of the paced hearts. CONCLUSION: The protective effect of chronic asynchronous pacing at near physiological rate on ventricular E-C coupling appears to be associated with subtle activation of L-type Ca(2+) channel, inhibition of sarcK(ATP) channel, and/or opening of mitoK(ATP) channel.

Potassium channel alterations mediate peripheral nerve hyperexcitability in a mouse model of type 2 diabetes mellitus

Diabetes mellitus (DM) is a major cause of peripheral neuropathy. More than 220 million people worldwide suffer from type 2 DM, which will, in approximately half of them, lead to the development of diabetic peripheral neuropathy. While of significant medical importance, the pathophysiological changes present in DPN are still poorly understood. To get more insight into DPN associated with type 2 DM, we decided to use the rodent model of this form of diabetes, the db/db mice. During the in-vivo conduction velocity studies on these animals, we observed the presence of multiple spiking followed by a single stimulation. This prompted us to evaluate the excitability properties of db/db peripheral nerves. Ex-vivo electrophysiological evaluation revealed a significant increase in the excitability of db/db sciatic nerves. While the shape and kinetics of the compound action potential of db/db nerves were the same as for control nerves, we observed an increase in the after-hyperpolarization phase (AHP) under diabetic conditions. Using pharmacological inhibitors we demonstrated that both the peripheral nerve hyperexcitability (PNH) and the increased AHP were mostly mediated by the decreased activity of Kv1-channels. Importantly, we corroborated these data at the molecular level. We observed a strong reduction of Kv1.2 channel presence in the juxtaparanodal regions of teased fibers in db/db mice as compared to control mice. Quantification of the amount of both Kv1.2 isoforms in DRG neurons and in the endoneurial compartment of peripheral nerve by Western blotting revealed that less mature Kv1.2 was integrated into the axonal membranes at the juxtaparanodes. Our observation that peripheral nerve hyperexcitability present in db/db mice is at least in part a consequence of changes in potassium channel distribution suggests that the same mechanism also mediates PNH in diabetic patients. ∗Current address: Department of Physiology, UCSF, San Francisco, CA, USA.

An exploratory study on facial emotion recognition capacity in beginning Alzheimer's disease.

Objective: It was the aim of this study to investigate facial emotion recognition (FER) in the elderly with cognitive impairment. Method: Twelve patients with Alzheimer's disease (AD) and 12 healthy control subjects were asked to name dynamic or static pictures of basic facial emotions using the Multimodal Emotion Recognition Test and to assess the degree of their difficulty in the recognition task, while their electrodermal conductance was registered as an unconscious processing measure. Results: AD patients had lower objective recognition performances for disgust and fear, but only disgust was accompanied by decreased subjective FER in AD patients. The electrodermal response was similar in all groups. No significant effect of dynamic versus static emotion presentation on FER was found. Conclusion: Selective impairment in recognizing facial expressions of disgust and fear may indicate a nonlinear decline in FER capacity with increasing cognitive impairment and result from progressive though specific damage to neural structures engaged in emotional processing and facial emotion identification. Although our results suggest unchanged unconscious FER processing with increasing cognitive impairment, further investigations on unconscious FER and self-awareness of FER capacity in neurodegenerative disorders are required.

Epineurial adipocytes are dispensable for Schwann cell myelination.

Previous clinical observations and data from mouse models with defects in lipid metabolism suggested that epineurial adipocytes may play a role in peripheral nervous system myelination. We have used adipocyte-specific Lpin1 knockout mice to characterize the consequences of the presence of impaired epineurial adipocytes on the myelinating peripheral nerve. Our data revealed that the capacity of Schwann cells to establish myelin, and the functional properties of peripheral nerves, were not affected by compromised epineurial adipocytes in adipocyte-specific Lpin1 knockout mice. To evaluate the possibility that Lpin1-negative adipocytes are still able to support endoneurial Schwann cells, we also characterized sciatic nerves from mice carrying epiblast-specific deletion of peroxisome proliferator-activated receptor gamma, which develop general lipoatrophy. Interestingly, even the complete loss of adipocytes in the epineurium of peroxisome proliferator-activated receptor gamma knockout mice did not lead to detectable defects in Schwann cell myelination. However, probably as a consequence of their hyperglycemia, these mice have reduced nerve conduction velocity, thus mimicking the phenotype observed under diabetic condition. Together, our data indicate that while adipocytes, as regulators of lipid and glucose homeostasis, play a role in nerve function, their presence in epineurium is not essential for establishment or maintenance of proper myelin.

Simulation des caractéristiques ECG du syndrome de Brugada à l'aide d'un programme de modélisation 3D

Introduction : Le syndrome de Brugada, décrit en 1992 par Pedro et Josep Brugada, est un syndrome cardiaque caractérisé par un sus-décalage particulier du segment ST associé à un bloc de branche droit atypique au niveau des dérivations ECG V1 à V3. Les altérations ECG du syndrome de Brugada sont classifiées en 3 types dont seul le type 1 est diagnostique. Les mécanismes physiopathologiques exacts de ce syndrome sont pour le moment encore controversés. Plusieurs hypothèses sont proposées dans la littérature dont deux principales retiennent l'attention : 1) le modèle du trouble de repolarisation stipule des potentiels d'action réduits en durée et en amplitude liés à un changement de répartition de canaux potassiques 2) le modèle du trouble de dépolarisation spécifie un retard de conduction se traduisant par une dépolarisation retardée. Dans le STEMI, un sus-décalage ST ressemblant à celui du syndrome de Brugada est expliqué par deux théories : 1) le courant de lésion diastolique suggère une élévation du potentiel diastolique transformé artificiellement en sus-décalage ST par les filtres utilisés dans tous les appareils ECG.¦Objectif : Recréer les manifestations ECG du syndrome de Brugada en appliquant les modifications du potentiel d'action des cardiomyocytes rapportées dans la littérature.¦Méthode : Pour ce travail, nous avons utilisé "ECGsim", un simulateur informatique réaliste d'ECG disponible gratuitement sur www.ecgsim.org. Ce programme est basé sur une reconstruction de l'ECG de surface à l'aide de 1500 noeuds représentant chacun les potentiels d'action des ventricules droit et gauche, épicardiques et endocardiques. L'ECG simulé peut être donc vu comme l'intégration de l'ensemble de ces potentiels d'action en tenant compte des propriétés de conductivité des tissus s'interposant entre les électrodes de surface et le coeur. Dans ce programme, nous avons définit trois zones, de taille différente, comprenant la chambre de chasse du ventricule droit. Pour chaque zone, nous avons reproduit les modifications des potentiels d'action citées dans les modèles du trouble de repolarisation et de dépolarisation et des théories de courant de lésion systolique et diastolique. Nous avons utilisé, en plus des douze dérivations habituelles, une électrode positionnée en V2IC3 (i.e. 3ème espace intercostal) sur le thorax virtuel du programme ECGsim.¦Résultats : Pour des raisons techniques, le modèle du trouble de repolarisation n'a pas pu être entièrement réalisée dans ce travail. Le modèle du trouble de dépolarisation ne reproduit pas d'altération de type Brugada mais un bloc de branche droit plus ou moins complet. Le courant de lésion diastolique permet d'obtenir un sus-décalage ST en augmentant le potentiel diastolique épicardique des cardiomyocytes de la chambre de chasse du ventricule droit. Une inversion de l'onde T apparaît lorsque la durée du potentiel d'action est prolongée. L'amplitude du sus-décalage ST dépend de la valeur du potentiel diastolique, de la taille de la lésion et de sa localisation épicardique ou transmurale. Le courant de lésion systolique n'entraîne pas de sus-décalage ST mais accentue l'amplitude de l'onde T.¦Discussion et conclusion : Dans ce travail, l'élévation du potentiel diastolique avec un prolongement de la durée du potentiel d'action est la combinaison qui reproduit le mieux les altérations ECG du Brugada. Une persistance de cellules de type nodal au niveau de la chambre de chasse du ventricule droit pourrait être une explication à ces modifications particulières du potentiel d'action. Le risque d'arythmie dans la Brugada pourrait également être expliqué par une automaticité anormale des cellules de type nodal. Ainsi, des altérations des mécanismes cellulaires impliqués dans le maintien du potentiel diastolique pourraient être présentes dans le syndrome de Brugada, ce qui, à notre connaissance, n'a jamais été rapporté dans la littérature.

Substitution of (R,S)-methadone by (R)-methadone: Impact on QTc interval.

Methadone is administered as a chiral mixture of (R,S)-methadone. The opioid effect is mainly mediated by (R)-methadone, whereas (S)-methadone blocks the human ether-à-go-go-related gene (hERG) voltage-gated potassium channel more potently, which can cause drug-induced long QT syndrome, leading to potentially lethal ventricular tachyarrhythmias. To investigate whether substitution of (R,S)-methadone by (R)-methadone could reduce the corrected QT (QTc) interval, (R,S)-methadone was replaced by (R)-methadone (half-dose) in 39 opioid-dependent patients receiving maintenance treatment for 14 days. (R)-methadone was then replaced by the initial dose of (R,S)-methadone for 14 days (n = 29). Trough (R)-methadone and (S)-methadone plasma levels and electrocardiogram measurements were taken. The Fridericia-corrected QT (QTcF) interval decreased when (R,S)-methadone was replaced by a half-dose of (R)-methadone; the median (interquartile range [IQR]) values were 423 (398-440) milliseconds (ms) and 412 (395-431) ms (P = .06) at days 0 and 14, respectively. Using a univariate mixed-effect linear model, the QTcF value decreased by a mean of -3.9 ms (95% confidence interval [CI], -7.7 to -0.2) per week (P = .04). The QTcF value increased when (R)-methadone was replaced by the initial dose of (R,S)-methadone for 14 days; median (IQR) values were 424 (398-436) ms and 424 (412-443) ms (P = .01) at days 14 and 28, respectively. The univariate model showed that the QTcF value increased by a mean of 4.7 ms (95% CI, 1.3-8.1) per week (P = .006). Substitution of (R,S)-methadone by (R)-methadone reduces the QTc interval value. A safer cardiac profile of (R)-methadone is in agreement with previous in vitro and pharmacogenetic studies. If the present results are confirmed by larger studies, (R)-methadone should be prescribed instead of (R,S)-methadone to reduce the risk of cardiac toxic effects and sudden death.

Developing clinical indication for multisite pacing.

The artificial activation of the heart modifies the mechanics of contraction and relaxation. While only little basic research has been addressed to this question, clinical observations showed that for hypertrophic as well as dilated cardiomyopathies appropriate pacing techniques can be useful. Pacing can influence the activation sequence. The spread out from a single site is slow, and so hypercontractility deminshed. With the use of multiple electrodes, two atrial and/or two ventricular, conduction delays in the atria or ventricles can be eliminated. Synchronisation of the cardiac activation has an anti-arrhythmic and positiv inotropic effect. This may lead to new indications for pacemakers or better to be named cardiac synchronisers.

A study of the cambial zone and conductive phloem of common beech (Fagus sylvatica L.) using an image analysis method. I. Influence of tree age on the structure

Quantification is a major problem when using histology to study the influence of ecological factors on tree structure. This paper presents a method to prepare and to analyse transverse sections of cambial zone and of conductive phloem in bark samples. The following paper (II) presents the automated measurement procedure. Part I here describes and discusses the preparation method, and the influence of tree age on the observed structure. Highly contrasted images of samples extracted at breast height during dormancy were analysed with an automatic image analyser. Between three young (38 years) and three old (147 years) trees, age-related differences were identified by size and shape parameters, at both cell and tissue levels. In the cambial zone, older trees had larger and more rectangular fusiform initials. In the phloem, sieve tubes were also larger, but their shape did not change and the area for sap conduction was similar in both categories. Nevertheless, alterations were limited, and demanded statistical analysis to be identified and ascertained. The physiological implications of the structural changes are discussed.

Altered distribution of juxtaparanodal kv1.2 subunits mediates peripheral nerve hyperexcitability in type 2 diabetes mellitus.

Peripheral nerve hyperexcitability (PNH) is one of the distal peripheral neuropathy phenotypes often present in patients affected by type 2 diabetes mellitus (T2DM). Through in vivo and ex vivo electrophysiological recordings in db/db mice, a model of T2DM, we observed that, in addition to reduced nerve conduction velocity, db/db mice also develop PNH. By using pharmacological inhibitors, we demonstrated that the PNH is mediated by the decreased activity of K(v)1-channels. In agreement with these data, we observed that the diabetic condition led to a reduced presence of the K(v)1.2-subunits in juxtaparanodal regions of peripheral nerves in db/db mice and in nerve biopsies from T2DM patients. Together, these observations indicate that the T2DM condition leads to potassium channel-mediated PNH, thus identifying them as a potential drug target to treat some of the DPN related symptoms.

Arrhythmogenesis in the developing heart during anoxia-reoxygenation and hypothermia-rewarming: an in vitro model.

INTRODUCTION: The spatio-temporal pattern of arrhythmias in the embryonic/fetal heart subjected to a transient hypoxic or hypothermic stress remains to be established. METHODS AND RESULTS: Spontaneously beating hearts or isolated atria, ventricles, and conotruncus from 4-day-old chick embryos were subjected in vitro to 30-minute anoxia and 60-minute reoxygenation. Hearts were also submitted to 30-minute hypothermia (0-4 degrees C) and 60-minute rewarming. ECG disturbances and alterations of atrial and ventricular electromechanical delay (EMD) were systematically investigated. Baseline functional parameters were stable during at least 2 hours. Anoxia induced tachycardia, followed by bradycardia, atrial ectopy, first-, second-, and third-degree atrio-ventricular blocks and, finally, transient electromechanical arrest after 6.8 minutes, interquartile ranges (IQR) 3.1-16.2 (n = 8). Reoxygenation triggered also Wenckebach phenomenon and ventricular escape beats. At the onset of reoxygenation QT, PR, and ventricular EMD increased by 68%, 70%, and 250%, respectively, whereas atrial EMD was not altered. No fibrillations, no ventricular ectopic beats, and no electromechanical dissociation were observed. Arrhythmic activity of the isolated atria persisted throughout anoxia and upon reoxygenation, whereas activity of the isolated ventricles abruptly ceased after 5 minutes of anoxia and resumed after 5 minutes of reoxygenation. During hypothermia-rewarming, cardiac activity stopped at 17.9 degrees C, IQR 16.2-20.6 (n = 4) and resumed at the same temperature with no arrhythmias. All preparations fully recovered after 40 minutes of reoxygenation or rewarming. CONCLUSION: In the embryonic heart, arrhythmias mainly originated in the sinoatrial tissue and resembled those observed in the adult heart. Furthermore, oxygen readmission was by far more arrhythmogenic than rewarming and the chronotropic, dromotropic, and inotropic effects were fully reversible.

Electrocardiographic findings in a middle-aged African population in the Seychelles islands.

This study describes major electrocardiogram (ECG) measurements and diagnoses in a population of African individuals; most reference data have been collected in Caucasian populations and evidence exists for interethnic differences in ECG findings. This study was conducted in the Seychelles islands (Indian Ocean) and included 709 black individuals (343 men and 366 women) aged 25 to 64 years randomly selected from the general population. Resting ECG were recorded by using a validated ECG unit equipped with a measurement and interpretation software (Cardiovit AT-6, Schiller, Switzerland). The epidemiology of 14 basic ECG measurements, 6 composite criteria for left ventricular hypertrophy and 19 specific ECG diagnoses including abnormal rhythms, conduction abnormalities, repolarization abnormalities, and myocardial infarction were examined. Substantial gender and age differences were found for several ECG parameters. Moreover, tracings recorded in African individuals of the Seychelles differed from those collected similarly in Caucasian populations in many respects. For instance, heart rate was approximately 5 beats per minute lower in the African individuals than in selected Caucasian populations, prevalence of first degree atrio-ventricular block was especially high (4.8%), and the average Sokolow-Lyon voltage was markedly higher in African individuals of the Seychelles compared with black and white Americans. The integrated interpretation software detected "old myocardial infarction" in 3.8% of men and 0% of women and "old myocardial infarction possible" in 6.1% and 3%, respectively. Cardiac infarction injury scores are also provided. In conclusion, the study provides reference values for ECG findings in a specific population of people of African descent and stresses the need to systematically consider gender, age, and ethnicity when interpreting ECG tracings in individuals.