Nitric oxide modulates Na+, K+-ATPase activity through cyclic GMP pathway in proximal rat trachea

The present work demonstrated that nitric oxide (NO) modulates Na+, K+-ATPase activity in the proximal rat trachea. Sodium nitroprusside induced concentration-dependent (10-100 microM) stimulation in proximal trachea Na+, K+-ATPase activity. The effect was specific for Na+, K+-ATPase since Mg-ATPase activity was unaffected. This NO-donor changed neither Na+, K+-ATPase nor Mg-ATPase activity in the distal segment. The modulatory action on Na+, K+-ATPase induced by sodium nitroprusside was linked to an increase in nitrates/nitrites and cyclic GMP levels in proximal segments. Modulation of proximal Na+, K+-ATPase activity by sodium nitroprusside was mimicked by S-nitroso-N-acetylpenicillamine (100 microM) and 8-bromo-cyclic GMP (100 microM). Both sodium nitroprusside and 8-bromo-cyclic GMP effects on Na+, K+-ATPase activity of proximal segments of trachea were blocked by 2 microM of KT 5823 (a cyclic GMP-dependent protein kinase inhibitor), but not by 0.5 microM of KT 5720 (a cyclic AMP-dependent protein kinase inhibitor). Both kinase inhibitors decreased proximal Na+, K+-ATPase activity, but did not change Mg-ATPase activity. Okadaic acid (1 microM), a phosphatase-1 inhibitor, increased proximal Na+, K+-ATPase but not Mg-ATPase activity. The effect of okadaic acid was non-additive with that of 8-bromo-cGMP on Na+, K+-ATPase activity. Our results suggest that NO modulates proximal rat trachea Na+, K+-ATPase activity through cyclic GMP and cyclic GMP-dependent protein kinase.     

Bone marrow micrometastases in 109 breast cancer patients: Correlations with clinical and pathological features and prognosis

Background: The presence in bone marrow of cells which react with monoclonal antibodies against tumor-associated antigens has been proposed over the last few years as a new prognostic factor in breast cancer patients. Patients and methods: Bone marrow aspirates were obtained from 109 stage I and II breast cancer patients during or 2–4 weeks after primary surgery. The samples were processed for leukocyte separation on a Ficoll-Hypaque gradient and then used to prepare cytospin slides for immunocytochemical analysis. The slides were stained with a pool of monoclonal antibodies (MoAbs) which recognize tumor associated antigens, using the alkaline phosphatase anti-alkaline phosphatase method. The median follow-up was 36 months (range 15–62); 22 patients relapsed and 7 died. Results: Thirty-four of the 109 patients (31.1%) had MoAb positive bone marrow cells. The bone marrow was positive in 28/74 (37.9%) patients who had the aspirate taken during surgery and in 6/35 (17.1%) who had it taken after surgery (p = 0.055). No association was found between bone marrow positivity and tumour size, nodal status, menopausal status, estrogen receptor positivity or the proliferative index. No association was found between bone marrow and prognosis: the log-rank test was 0.291 (p > 0.5) for OS and 0.023 for DFS; the hazard ratio (positive vs negative) was 1.51 for OS (95% CI: 0.33–6.86) and 0.93 for DFS (95% CI: 0.35–2.45). Conclusions: In our series, bone marrow positivity did not correlate with prognostic parameters or prognosis. Of interest is the relative excess of positivity when the bone marrow was obtained during surgery.     

Myofiberbreak-up: a marker of ventricular fibrillation in sudden cardiac death

BACKGROUND: Electrophysiologically, ventricular fibrillation is defined as a "chaotic, random, asynchronous electrical activity of the ventricles due to repetitive re-entrant excitation and/or rapid focal discharge". To this point its morphological equivalent has not been defined. MATERIAL AND METHOD: Several groups of different diseases and types of accidental death in normal subjects were studied. A complete autopsy was performed and the hearts were examined in 432 cases. A total of 16 myocardial samples per heart were processed for histological examination and sections were stained by haematoxylin and eosin or by specific stains. The frequency, location and extent of myocellular segmentation (stretching and/or rupture) of intercalated discs and associated changes of myocardial bundles and single myocells were investigated. A quantitative analysis was performed and the data were processed for statistical evaluation. RESULTS: The frequency of MFB was maximal in coronary (88%) and Chagas (76%) groups followed by the intracranial brain haemorrhage group (52%). The extent of myofiberbreak-up was maximal in coronary/Chagas groups followed by intracranial haemorrhage and transplant groups. CONCLUSIONS: No correlation was seen between gender, age, heart weight, degree of coronary atherosclerosis, myocardial fibrosis, survival and MFB. If our postulate is correct, finding MFB in the myocardium might allow the diagnosis of a malignant arrhythmia followed by cardiac arrest due to ventricular fibrillation even in the absence of clinical information (sudden death out-of-hospital).     

Allergic rhinitis phenotypes based on mono-allergy or poly-allergy

Inflammation Research - Allergic rhinitis (AR) is characterized by typical symptoms that are dependent on inflammation. Poly-allergy is a frequent phenomenon. Phenotyping AR represents an...     

Repression of Flagella Is a Common Trait in Field Isolates of Salmonella enterica Serovar Dublin and Is Associated with Invasive Human Infections

The nontyphoidal Salmonella enterica serovar Dublin is adapted to cattle but infrequently infects humans, very often resulting in invasive infections with high levels of morbidity and mortality. A Salmonella-induced intestinal acute inflammatory response is postulated as a mechanism to prevent bacterial dissemination to systemic sites. In S. enterica serovar Typhimurium, flagella contribute to this response by providing motility and FliC-mediated activation of pattern recognition receptors. In this study, we found 4 Salmonella enterica isolates, with the antigenic formula 9,12:−:−, that, based on fliC sequence and multilocus sequence type (MLST) analyses, are aflagellate S. Dublin isolates. Interestingly, all were obtained from human bloodstream infections. Thus, we investigated the potential role of flagella in the unusual invasiveness exhibited by S. Dublin in humans by analyzing flagellation and proinflammatory properties of a collection of 10 S. Dublin human clinical isolates. We found that 4 of 7 blood isolates were aflagellate due to significantly reduced levels of fliC expression, whereas all 3 isolates from other sources were flagellated. Lack of flagella correlated with a reduced ability of triggering interleukin-8 (IL-8) and CCL20 chemokine expression in human intestinal Caco-2 cells and with reduced early inflammation in the ceca of streptomycin-pretreated C57/BL6 mice. These results indicate that flagella contribute to the host intestinal inflammatory response to Salmonella serovar Dublin and suggest that their absence may contribute to its systemic dissemination through dampening of the gut immune response. Analysis of FliC production in a collection of cattle isolates indicated that the aflagellate phenotype is widely distributed in field isolates of S. Dublin.     

The endocannabinoid 2-AG controls skeletal muscle cell differentiation via CB1 receptor-dependent inhibition of Kv7 channels

Little is known of the involvement of endocannabinoids and cannabinoid receptors in skeletal muscle cell differentiation. We report that, due to changes in the expression of genes involved in its metabolism, the levels of the endocannabinoid 2-arachidonoylglycerol (2-AG) are decreased both during myotube formation in vitro from murine C₂C₁₂ myoblasts and during mouse muscle growth in vivo. The endocannabinoid, as well as the CB1 agonist arachidonoyl-2-chloroethylamide, prevent myotube formation in a manner antagonized by CB1 knockdown and by CB1 antagonists, which, per se, instead stimulate differentiation. Importantly, 2-AG also inhibits differentiation of primary human satellite cells. Muscle fascicles from CB1 knockout embryos contain more muscle fibers, and postnatal mice show muscle fibers of an increased diameter relative to wild-type littermates. Inhibition of K_v7.4 channel activity, which plays a permissive role in myogenesis and depends on phosphatidylinositol 4,5-bisphosphate (PIP2), underlies the effects of 2-AG. We find that CB1 stimulation reduces both total and K_v7.4-bound PIP2 levels in C₂C₁₂ cells and inhibits K_v7.4 currents in transfected CHO cells. We suggest that 2-AG is an endogenous repressor of myoblast differentiation via CB1-mediated inhibition of K_v7.4 channels.The endocannabinoid system (ECS) refers to a large group of endogenous molecules including the two major arachidonate-derived neuromodulatory mediators, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), known as endocannabinoids (EC); several enzymes involved in the metabolism of AEA (NAPE-PLD, ABDH4, GDE1, PTPN22 for biosynthesis and FAAH for degradation) and 2-AG (DAGLα and DAGLβ for biosynthesis and MAGL, ABDH6, ABDH12, and FAAH for degradation); and two G protein-coupled receptors known as cannabinoid receptor of type-1 (CB1) and type-2 (CB2). AEA also activates the cation permeant transient receptor potential vanilloid type-1 (TRPV1) channels (1). In mammals, the ECS regulates a large number of physiological processes; alterations in its activity are in fact responsible for the onset or progression of many types of disorders affecting both the central and the peripheral nervous system as well as other organs (2–5). So far, a few studies have reported that CB1 receptor activity controls key skeletal muscle metabolic processes such as insulin signaling, glucose uptake, and fatty acid oxidation (6, 7). However, little, if anything at all, is known about the expression profile and the functional role played by the ECS during skeletal muscle development.Skeletal myogenesis is a tightly regulated process that requires coordinated changes in a large number of genes allowing proliferating myoblasts to withdraw from the cell cycle and fuse to form large multinucleated myotubes (8). Several classes of ion channels play a pivotal role in the initiation of the differentiation process. For example, the sequential activation of two distinct classes of K⁺ channels, the ether-a-go-go K_v10.1 and the inward-rectifier KIR2.1 (9, 10), is known to be one of the first molecular events that causes myoblast hyperpolarization. This event, in turn, leads to the activation of voltage-dependent T-type Ca²⁺ channels, which increase the [Ca²⁺]_i necessary to initiate myoblast commitment to differentiation into myotubes (11). More recently, members of the K_v7 (KCNQ) subfamily of voltage-activated K⁺ channels have been found to be expressed in both myoblasts and myotubes (12, 13), and, in particular, it has been shown that K_v7.4 channel expression plays a permissive role in skeletal myogenesis (14).The K_v7 subfamily comprises five subunits (K_v7.1–K_v7.5), each showing distinct tissue distribution and physiological properties. K_v7 channel function is regulated by several classes of G_q/11-coupled receptors including muscarinic (15), bradikynin (16), serotonin (17), and somatostatin receptors (18). Stimulation of these receptors leads to phospholipase C (PLC) activation and subsequent hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). Thus, considering that PIP2 is strictly required for K_v7 channels activity, G_q/11-coupled receptor stimulation represents one of the most important cellular mechanisms through which this subclass of K⁺ channels is kept under negative control (19). Interestingly, the M current, which is underlied by K_v7 channels, can be also inhibited following CB1 receptor stimulation by AEA at the postsynaptic level in hippocampal neurons (20) or by stimulation of the G_q/11-coupled orphan receptor GPR55 (21).In this study, we have endeavored to understand the role played by the ECS in muscle development and its impact on K_v7 activity during myogenesis by using molecular biology, biochemical, pharmacological, morphological, and electrophysiological techniques. Our results indicate that the endocannabinoid 2-AG tonically inhibits differentiation of mouse and human myoblasts via sequential activation of CB1 receptors, reduction of PIP2 levels, and inhibition of K_v7 channel activity.