Exercise hyperventilation, dyspnea sensation, and ergoreflex activation in lone atrial fibrillation

Lone atrial fibrillation may be associated with daily life disability and exercise limitation. The extracardiac pathophysiology of these effects is poorly explored. In 35 subjects with lone atrial fibrillation (mean age 67 +/- 7 yr), we investigated pulmonary function, symptom-limited cardiopulmonary exercise performance, muscle ergoreflex (handgrip exercise) contribution to ventilation, and brachial artery flow-mediated dilation (as a measure of endothelial function) before and after (average interval 20 +/- 5 days) restoring sinus rhythm with external cardioversion. Respiratory volumes and lung diffusing capacity at rest were within normal limits during both atrial fibrillation and after restoring sinus rhythm. Cardioversion was associated with the following changes: a decrease of the slope of exercise ventilation vs. CO2 production (from 35 +/- 5 to 29 +/- 3; P <0.01) and of dyspnea sensation (Borg score from 4 to 2) and an increase of peak oxygen uptake (Vo2; from 16 +/- 4 to 20 +/- 5 ml.min(-1).kg(-1); P <0.01), Vo2 at anaerobic threshold (from 11 +/- 2 to 13 +/- 2 ml.min(-1).kg(-1); P <0.05), and O2 pulse (from 8 +/- 3 to 11 +/- 3 ml/beat; P <0.01). After cardioversion, the observed improvement in ventilatory efficiency was accompanied by a significant peak end-tidal CO2 increase (from 33 +/- 2 to 37 +/- 2 mmHg; P <0.01) and no changes in dead space-to-tidal volume ratio (from 0.23 +/- 0.03 to 0.23 +/- 0.02; P=not significant). In addition, the ergoreflex contribution to ventilation was remarkably attenuated, and the brachial artery flow-mediated dilatation was significantly augmented (from 0.32 +/- 0.07 to 0.42 +/- 0.08 mm; P <0.01). Ten patients had atrial fibrillation relapse and, compared with values after restoration of regular sinus rhythm, invariably showed worsening of endothelial function, exercise ventilatory efficiency, and muscle ergoreflex contribution to ventilation. In subjects with lone atrial fibrillation, an impairment in ventilatory efficiency appears to be involved in the pathophysiology of exercise limitation, and to be primarily related with a demodulated peripheral control of ventilation.     

Structural study of four-stranded quadruplex structures containing 2'-deoxy-8-(propyn-1-yl)adenosine

In this paper, we report the NMR structural study of two quadruplex structures formed by truncations of the human telomeric sequence and containing a modified base, namely d(AprGGGT) and d(TAprGGGT), where Apr indicates 2'-deoxy-8-(propyn-1-yl)adenosines. Both oligonucleotides have been found to form 4-fold symmetric G-quadruplex structures with all strands parallel and equivalent to each other and characterized by higher thermal stabilities than the natural counterparts. The presence of the propynyl groups affects the conformations of the 5' edge of both quadruplexes in such a way to prevent the formation of one of the two possible H-bond patterns observed for a canonical A-tetrad. The increased thermal stabilities of the modified quadruplexes seem to be mostly due to a prevalent syn glycosidic conformation assumed by the Apr residues.     

Reaction of peroxynitrite with hyaluronan and related saccharides

The effects of peroxynitrite on hyaluronan has been studied by using an integrated spectroscopical approach, namely electron paramagnetic resonance (EPR), nuclear magnetic resonance (NMR), and mass spectrometry (MS). The reaction has been performed with the polymer, the tetrasaccharide oligomer as well as with the monosaccharides N-acetylglucosamine and glucuronic acid. The outcome of the presence of molecular oxygen and carbon dioxide has been also evaluated. Although ₁H-NMR and ESI-MS experiments did not revealed peroxynitrite-mediated modification of hyaluronan as well as of related saccharides, from spin-trapping EPR experiments it was concluded that peroxynitrite induce the formation of C-centered carbon radicals, most probably by the way of its hydroxyl radical-like reactivity. These EPR data support the oxidative pathway involved in the degradation of hyaluronan, a probable event in the development and progression of rheumatoid arthritis.     

Modulation of endothelin-A receptor, Galpha subunit, and RGS2 expression during H9c2 cardiomyoblast differentiation

In cardiac myocytes, growth responses depend on activation of G protein-coupled receptors interacting with Gq/11 protein subfamily members. Endothelin receptors of the ETA subtype belong to this receptor group inducing hypertrophic responses. To understand the role of ETA receptors and signal transduction proteins in modulating cell growth, we analyzed the pharmacological profile of this receptor, its level of expression together with those of Galpha subunits and the RGS2 protein in cardiomyoblasts differentiating into the cardiac phenotype. H9c2 rat cardiomyoblasts were grown in the presence of 10% fetal bovine serum (FBS) or 1% FBS plus all-trans-retinoic acid to induce the cardiac phenotype. The pharmacological properties of ETA receptors were investigated by competition-binding experiments, whereas the protein expression profile was analyzed by immunoblot and immunocytochemistry. The pharmacological profile of ETA receptors changed during differentiation of cardiomyoblasts into cardiomyocytes, and the amount of expressed receptor appeared to increase. Immunocytochemistry also showed a marked increase of receptor expression on cell membranes of differentiated cardiomyocytes. Among the other signaling proteins examined, both Galphaq/11 and RGS2 expression decreased in cells with the cardiac phenotype. Our results demonstrate that the expression of key proteins (ETA receptor, Galphaq/11, and RGS2) involved in signal transduction of hypertrophic stimuli is modulated during cell differentiation and correlates with the cardiac phenotype.     

Glutamate 59 is critical for transport function of the amino acid cotransporter KAAT1

KAAT1 is a neutral amino acid transporter activated by K⁺ or by Na⁺ (9). The protein shows significant homology with members of the Na⁺/Cl^–-dependent neurotransmitter transporter super family. E59G KAAT1, expressed in Xenopus oocytes, exhibited a reduced leucine uptake [20–30% of wild-type (WT)], and kinetic analysis indicated that the loss of activity was due to reduction of V_max and apparent affinity for substrates. Electrophysiological analysis revealed that E59G KAAT1 has presteady-state and uncoupled currents larger than WT but no leucine-induced currents. Site-directed mutagenesis analysis showed the requirement of a negative charge in position 59 of KAAT1. The analysis of permeant and impermeant methanethiosulfonate reagent effects confirmed the intracellular localization of glutamate 59. Because the 2-aminoethyl methanethiosulfonate hydrobromid inhibition was not prevented by the presence of Na⁺ or leucine, we concluded that E59 is not directly involved in the binding of substrates. N-ethylmaleimide inhibition was qualitatively and quantitatively different in the two transporters, WT and E59G KAAT1, having the same cysteine residues. This indicates an altered accessibility of native cysteine residues due to a modified spatial organization of E59G KAAT1. The arginine modifier phenylglyoxal effect supports this hypothesis: not only cysteine but also arginine residues become more accessible to the modifying reagents in the mutant E59G. In conclusion, the results presented indicate that glutamate 59 plays a critical role in the three-dimensional organization of KAAT1. amino acid transport; structure/function; amino acid modifiers; Manduca sexta     

Preimaginal learning determines adult response to chemical stimuli in a parasitic wasp

The behavioural responses of parasitic wasps to chemical cues from their hosts and host plants are known to be affected by genetic and environmental components. In a previous study of the codling moth ectoparasitoid Hyssopus pallidus, we found that the response of adult parasitoids to the frass of their host caterpillars depended on a learning process involving plant cues. In the present study, we investigated how and when learning takes place. A series of experiments was conducted involving exposure of parasitoids to fruit cues at different developmental stages. While parasitoids were not able to learn the fruit cues in the adult stage, exposure to fruit odour at early preimaginal stages significantly increased the adult response to frass from fruit-fed caterpillars. The olfactory memory persisted through metamorphosis, with a retention time of 14 days. Preimaginal learning was not confined to fruit cues but was also demonstrated for a host- and fruit-independent cue, menthol. Parasitoids exposed to menthol odour at the egg and larval stages no longer showed negative responses as adults. Sensitization to fruit cues and habituation to menthol are considered to be the mechanisms involved. This study provides evidence of true preimaginal learning of olfactory cues in a parasitic wasp.     

Abnormal splicing of ABCA1 pre-mRNA in Tangier disease due to a IVS2 +5G>C mutation in ABCA1 gene

Two point mutations of ABCA1 gene were found in a patient with Tangier disease (TD): i) G>C in intron 2 (IVS2 +5G>C) and ii) c.844 C>T in exon 9 (R282X). The IVS2 +5G>C mutation was also found in the brother of another deceased TD patient, but not in 78 controls and 33 subjects with low HDL. The IVS2 +5G>C mutation disrupts ABCA1 pre-mRNA splicing in fibroblasts, leading to three abnormal mRNAs: devoid of exon 2 (Ex2-/mRNA), exon 4 (Ex4-/mRNA), or both these exons (Ex2-/Ex4-/mRNA), each containing a translation initiation site. These mRNAs are expected either not to be translated or generate short peptides. To investigate the in vitro effect of IVS2 +5G>C mutation, we constructed two ABCA1 minigenes encompassing Ex1-Ex3 region, one with wild-type (WTgene) and the other with mutant (MTgene) intron 2. These minigenes were transfected into COS1 and NIH3T3, two cell lines with a different ABCA1 gene expression. In COS1 cells, WTgene pre-mRNA was spliced correctly, while the splicing of MTgene pre-mRNA resulted in Ex2-/mRNA. In NIH3T3, no splicing of MTgene pre-mRNA was observed, whereas WTgene pre-mRNA was spliced correctly. These results stress the complexity of ABCA1 pre-mRNA splicing in the presence of splice site mutations.     

Leber's hereditary optic neuropathy (LHON) pathogenic mutations induce mitochondrial-dependent apoptotic death in transmitochondrial cells incubated with galactose medium

Leber's hereditary optic neuropathy (LHON), a maternally inherited form of central vision loss, is associated with mitochondrial DNA pathogenic point mutations affecting different subunits of complex I. We here report that osteosarcoma-derived cytoplasmic hybrids (cybrid) cell lines harboring one of the three most frequent LHON pathogenic mutations, at positions 11778/ND4, 3460/ND1, and 14484/ND6, undergo cell death when galactose replaces glucose in the medium, contrary to control cybrids that maintain some growth capabilities. This is a well known way to produce a metabolic stress, forcing the cells to rely on the mitochondrial respiratory chain to produce ATP. We demonstrate that LHON cybrid cell death is apoptotic, showing chromatin condensation and nuclear DNA laddering. Moreover, we also document the mitochondrial involvement in the activation of the apoptotic cascade, as shown by the increased release of cytochrome c into the cytosol in LHON cybrid cells as compared with controls. Cybrids bearing the 3460/ND1 and 14484/ND6 mutations seemed more readily prone to undergo apoptosis as compared with the 11778/ND4 mutation. In conclusion, LHON cybrid cells forced by the reduced rate of glycolytic flux to utilize oxidative metabolism are sensitized to an apoptotic death through a mechanism involving mitochondria.