Clinical and biological investigation of NO

Furchgott et al. demonstrated in 1980 that relaxation of arterial smooth muscle cells in response to acetylcholine is dependent on the integrity of endothelium. They named the factor responsible of this intercellular relationship EDRF (Endothelium Derived Relaxing Factor), which was identified 7 years latter as nitric oxide (NO), a free radical gas. In vessels, NO is generated locally by the endothelial NO synthase and its effect is mainly paracrine (relaxation of the underlying smooth muscle cells, and inhibition of platelet aggregation). The in vivo half-life of NO is short, and the assessment of its production is thus difficult. Invasive and non invasive techniques are now available to explore the variations of arterial diameter or flow. Furchgott's pioneering work anticipated the whole pathophysiology of endothelial-dependent relaxation. Indeed, numerous diseases, in particular atherosclerosis, are accompanied by abnormalities of endothelial-dependent vasodilation ("endothelial dysfunction"). Whereas acetylcholine (or serotonin) infused in a normal artery elicits a vasodilation, in contrast, it promotes a vasoconstriction in an atheromatous artery, as a consequence of a decrease in NO bioavailability. This defect in NO favors arterial spasm, interaction between platelets and arterial wall and thrombosis, and thus probably cardiovascular events. NO cannot be measured directly in humans, except in exhaled NO. In vivo, NO is rapidly oxidized in nitrite (NO2-) and in nitrate (NO3-), the summation being NOx. We shall detail the limitations of this measurement as a biochemical index of NO production from "endothelial" origin.     

Quantitative trait loci for grain quality, productivity, morphological and agronomical traits in sorghum (Sorghum bicolor L. Moench)

 Quantitative trait loci (QTLs) for grain quality, yield components and other traits were investigated in two Sorghum caudatum×guinea recombinant inbred line (RIL) populations. A total of 16 traits were evaluated (plant height, panicle length, panicle compactness, number of kernels/panicle, thousand-kernel weight, kernel weight/panicle, threshing percentage, dehulling yield, kernel flouriness, kernel friability, kernel hardness, amylose content, protein content, lipid content, germination rate and molds during germination and after harvest) and related to two 113- and 100-point base genetic maps using simple (SIM) and composite (CIM) interval mapping. The number, effects and relative position of QTLs detected in both populations were generally in agreement with the distributions, heritabilities and correlations among traits. Several chromosomal segments markedly affected multiple traits and were suspected of harbouring major genes. The positions of these QTLs are discussed in relation to previously reported studies on sorghum and other grasses. Many QTLs, depending on their relative effects and position, could be used as targets for marker-assisted selection and provide an opportunity for accelerating breeding programmes. Received: 14 February 1998 / Accepted: 4 March 1998     

The protective effect of the vagus nerve in a murine model of chronic relapsing colitis

The vagus nerve inhibits the response to systemic administration of endotoxin, and we have recently extended this observation to show that the vagus attenuates acute experimental colitis in mice. The purpose of the present study was to determine whether there is a tonic counterinflammatory influence of the vagus on colitis maintained over several weeks. We assessed disease activity index, macroscopic and histological damage, myeloperoxidase (MPO) activity, and Th1 and Th2 cytokine profiles in chronic colitis induced by administration of dextran sodium sulfate (DSS) in drinking water for three cycles during 5 days with 11 days of rest between each cycle (DSS 3, 2, 2%) in healthy and vagotomized C57BL/6 mice and in mice deficient in macrophage-colony stimulating factor (M-CSF). A pyloroplasty was performed in vagotomized mice. Vagotomy accelerated the onset and the severity of inflammation during the first and second but not the third cycle. Although macroscopic scores were not significantly changed, histological scores as well as MPO activity and colonic tissue levels of IL-1alpha, TNF-alpha, IFN-gamma, and IL-18 but not IL-4 were significantly increased in vagotomized mice compared with sham-operated mice that received DSS. In control mice (without colitis), vagotomy per se did not affect any inflammatory marker. Vagotomy had no effect on the colitis in M-CSF-derived macrophage-deficient mice. These results indicate that the vagus protects against acute relapses on a background of chronic inflammation. Identification of the molecular mechanisms underlying the protective role of parasympathetic nerves opens a new therapeutic avenue for the treatment of acute relapses of chronic inflammatory bowel disease.     

Characterization of articular cartilage by combining microscopic analysis with a fibril-reinforced finite-element model

Load-bearing characteristics of articular cartilage are impaired during tissue degeneration. Quantitative microscopy enables in vitro investigation of cartilage structure but determination of tissue functional properties necessitates experimental mechanical testing. The fibril-reinforced poroviscoelastic (FRPVE) model has been used successfully for estimation of cartilage mechanical properties. The model includes realistic collagen network architecture, as shown by microscopic imaging techniques. The aim of the present study was to investigate the relationships between the cartilage proteoglycan (PG) and collagen content as assessed by quantitative microscopic findings, and model-based mechanical parameters of the tissue. Site-specific variation of the collagen network moduli, PG matrix modulus and permeability was analyzed. Cylindrical cartilage samples (n=22) were harvested from various sites of the bovine knee and shoulder joints. Collagen orientation, as quantitated by polarized light microscopy, was incorporated into the finite-element model. Stepwise stress-relaxation experiments in unconfined compression were conducted for the samples, and sample-specific models were fitted to the experimental data in order to determine values of the model parameters. For comparison, Fourier transform infrared imaging and digital densitometry were used for the determination of collagen and PG content in the same samples, respectively. The initial and strain-dependent fibril network moduli as well as the initial permeability correlated significantly with the tissue collagen content. The equilibrium Young's modulus of the nonfibrillar matrix and the strain dependency of permeability were significantly associated with the tissue PG content. The present study demonstrates that modern quantitative microscopic methods in combination with the FRPVE model are feasible methods to characterize the structure-function relationships of articular cartilage.     

The first genomewide interaction and locus-heterogeneity linkage scan in bipolar affective disorder: strong evidence of epistatic effects between loci on chromosomes 2q and 6q

We present the first genomewide interaction and locus-heterogeneity linkage scan in bipolar affective disorder (BPAD), using a large linkage data set (52 families of European descent; 448 participants and 259 affected individuals). Our results provide the strongest interaction evidence between BPAD genes on chromosomes 2q22-q24 and 6q23-q24, which was observed symmetrically in both directions (nonparametric LOD [NPL] scores of 7.55 on 2q and 7.63 on 6q; P<.0001 and P=.0001, respectively, after a genomewide permutation procedure). The second-best BPAD interaction evidence was observed between chromosomes 2q22-q24 and 15q26. Here, we also observed a symmetrical interaction (NPL scores of 6.26 on 2q and 4.59 on 15q; P=.0057 and .0022, respectively). We covered the implicated regions by genotyping additional marker sets and performed a detailed interaction linkage analysis, which narrowed the susceptibility intervals. Although the heterogeneity analysis produced less impressive results (highest NPL score of 3.32) and a less consistent picture, we achieved evidence of locus heterogeneity at chromosomes 2q, 6p, 11p, 13q, and 22q, which was supported by adjacent markers within each region and by previously reported BPAD linkage findings. Our results provide systematic insights in the framework of BPAD epistasis and locus heterogeneity, which should facilitate gene identification by the use of more-comprehensive cloning strategies.     

The serine protease Omi/HtrA2 is involved in XIAP cleavage and in neuronal cell death following focal cerebral ischemia/reperfusion

Omi/HtrA2 is a pro-apoptotic mitochondrial serine protease involved in both forms of apoptosis, caspase-dependent as well as caspase-independent cell death. However, the impact of Omi/HtrA2 in the apoptotic cell machinery that takes place in vivo under pathological conditions such as cerebral ischemia remains unknown. The present study was monitored in order to examine whether Omi/HtrA2 plays a decisive role in apoptosis observed after focal cerebral ischemia in rats. Male adult rats were subjected to 90min of focal cerebral ischemia followed by reperfusion and treated with vehicle or ucf-101, a novel and specific Omi/HtrA2 inhibitor, prior reperfusion. Focal cerebral ischemia/reperfusion induced a mitochondrial up-regulation of Omi/HtrA2 and significantly increased cytosolic accumulation of Omi/HtrA2. Furthermore, ischemia led to activation of caspase-3 and degradation X-linked inhibitor of apoptosis protein (XIAP). Treatment of animals prior ischemia with ucf-101, the specific inhibitor of Omi/HtrA2, was able to (1) reduce the number of TUNEL-positive cells, to (2) attenuate the XIAP-breakdown and to (3) reduce the infarct size. This study shows for the first time that focal cerebral ischemia in rats results in Omi/HtrA2 translocation from the mitochondria to the cytosol, where it participates in neuronal cell death. Blocking the proteolytic activity of Omi/HtrA2 with specific inhibitors, such as the ucf-101, could be a novel way to afford neuroprotection and minimize cellular damage in cerebral ischemia/reperfusion.     

Endosomal receptor kinetics determine the stability of intracellular growth factor signalling complexes

There is an emerging paradigm that growth factor signalling continues in the endosome and that cell response to a growth factor is defined by the integration of cell surface and endosomal events. As activated receptors in the endosome are exposed to a different set of binding partners, they probably elicit differential signals compared with when they are at the cell surface. As such, complete appreciation of growth factor signalling requires understanding of growth factor-receptor binding and trafficking kinetics both at the cell surface and in endosomes. Growth factor binding to surface receptors is well characterized, and endosomal binding is assumed to follow surface kinetics if one accounts for changes in pH. Yet, specific binding kinetics within the endosome has not been examined in detail. To parse the factors governing the binding state of endosomal receptors we analysed a whole-cell mathematical model of epidermal growth factor receptor trafficking and binding. We discovered that the stability of growth factor-receptor complexes within endosomes is governed by three primary independent factors: the endosomal dissociation constant, total endosomal volume and the number of endosomal receptors. These factors were combined into a single dimensionless parameter that determines the endosomal binding state of the growth factor-receptor complex and can distinguish different growth factors from each other and different cell states. Our findings indicate that growth factor binding within endosomal compartments cannot be appreciated solely on the basis of the pH-dependence of the dissociation constant and that the concentration of receptors in the endosomal compartment must also be considered.     

Downregulation of connexin40 and increased prevalence of atrial arrhythmias in transgenic mice with cardiac-restricted overexpression of tumor necrosis factor

Atrial arrhythmias, primarily atrial fibrillation, have been independently associated with structural remodeling and with inflammation. We hypothesized that sustained inflammatory signaling by tumor necrosis factor (TNF) would lead to alterations both in underlying atrial myocardial structure and in atrial electrical conduction. We performed ECG recording, intracardiac electrophysiology studies, epicardial mapping, and connexin immunohistochemical analyses on transgenic mice with targeted overexpression of TNF in the cardiac compartment (MHCsTNF) and on wild-type (WT) control mice (age 8-16 wk). Atrial and ventricular conduction abnormalities were always evident on ECG in MHCsTNF mice, including a shortened atrioventricular interval with a wide QRS duration secondary to junctional rhythm. Supraventricular arrhythmias were observed in five of eight MHCsTNF mice, whereas none of the mice demonstrated ventricular arrhythmias. No arrhythmias were observed in WT mice. Left ventricular conduction velocity during apical pacing was similar between the two mouse groups. Connexin40 was significantly downregulated in MHCsTNF mice. In contrast, connexin43 density was not significantly altered in MHCsTNF mice, but rather dispersed away from the intercalated disks. In conclusion, sustained inflammatory signaling contributed to atrial structural remodeling and downregulation of connexin40 that was associated with an increased prevalence of atrial arrhythmias.     

Structure-activity relationship (SAR) investigations of substituted imidazole analogs as TRPV1 antagonists

A novel series of 4,5-biarylimidazoles as TRPV1 antagonists were designed based on the previously reported 4,6-disubstituted benzimidazole series. The analogs were evaluated for their ability to block capsaicin- or acid-induced calcium influx in TRPV1-expressing CHO cells. These studies led to the identification of a highly potent and orally bioavailable TRPV1 antagonist, imidazole 33.