BMPR2 mutations and response to inhaled or parenteral prostanoids: a case series.

Whether mutations in the BMPR2 gene may influence the response to PAH-specific therapies has not yet been investigated. In this study, in 13 idiopathic, heritable or anorexigen-associated PAH patients, in whom treatment escalation was performed by adding a prostanoid, a greater haemodynamic improvement was observed in BMPR2-negative than in BMPR2-positive patients.

Helicobacter pylori L-asparaginase: A Novel Bacterial Antigen that May Contribute to Infection Detection.

Helicobacter pylori is responsible for gastric inflammation and for an increased risk of cancer development in humans. Several bacterial antigens contribute to stimulate the immune system, but their relative role has not yet been defined. H. pylori (strain CCUG) type II L-asparaginase (L-ASNase) induces an immune response in mice. To verify if an immune response could also be detected in humans, sera positive (n=11) or negative (n=11), respectively, to H. pylori according to a commercial test were assayed for their reactivity towards purified H. pylori L-ASNase. Among positive samples, 8/11 (72%) were positive to L-ASNase. We conclude that H. pylori L-ASNase is immunogenic in humans and contributes to the generation of the antibody response induced by the bacterium.

Different forms of pulmonary hypertension in a family with clinical and genetic evidence for hereditary hemorrhagic teleangectasia type 2.

Hereditary hemorrhagic telangiectasia (HTT) is an autosomal dominant disease, most frequently caused by a mutation in either ENG or ACVRL1, which can be associated with pulmonary arterial hypertension (PAH). In this report, we describe a new unpublished ACVRL1 mutation segregating in three members of the same family, showing three different types of pulmonary hypertension (PH) in the absence of BMPR2 mutations. The first patient has a form of heritable PAH (HPAH) in the absence of hepatic arteriovenous malformations (AVMs); the second one has a severe form of portopulmonary hypertension (PoPAH) associated with multiple hepatic AVMs; the third one has hepatopulmonary syndrome (HPS) with numerous hepatic arteriovenous fistulas and a form of post-capillary PH due to high cardiac output. In summary, a single mutation in the ACVRL1 gene can be associated, in the same family, with an extreme phenotypic variability regarding not only the clinical presentation of HHT but also the type of PH in the absence of BMPR2 mutations. More studies are needed to evaluate if this variability can be explained by the presence of additional variants in other genes relevant for the pathogenesis of HHT.

Functional analysis of a novel ENG variant in a patient with hereditary hemorrhagic telangiectasia (HHT) identifies a new Sp1 binding-site.

Hereditary Hemorrhagic Telangiectasia (HHT) is a rare disease, with an autosomal dominant inheritance and a worldwide incidence of about 1: 5000 individuals. In >80% of patients, HHT is caused by mutations in either ENG or ACVRL1, which code for ENDOGLIN and Activin A Receptor Type II-Like Kinase 1 (ALK1), belonging to the TGF-ß/BMP signalling pathway. Typical HHT clinical features are mucocutaneous telangiectases, arteriovenous malformations, spontaneous and recurrent epistaxis, as well as gastrointestinal bleedings. An additional, but less frequent, clinical manifestation in some HHT patients is the presence of Pulmonary Arterial Hypertension (PAH). The aim of this work is to describe the functional role of a novel ENG intronic variant found in a patient affected by both HHT and PAH, in order to assess whether it has a pathogenic role. We proved that the variant lies in a novel binding-site for the transcription factor Sp1, known to be involved in the regulation of ENG and ACVRL1 transcription. We confirmed a pathogenic role for this intronic variant, as it significantly reduces ENG transcription by affecting this novel Sp1 binding-site.

Cell-cycle inhibition by Helicobacter pylori L-asparaginase.

Helicobacter pylori (H. pylori) is a major human pathogen causing chronic gastritis, peptic ulcer, gastric cancer, and mucosa-associated lymphoid tissue lymphoma. One of the mechanisms whereby it induces damage depends on its interference with proliferation of host tissues. We here describe the discovery of a novel bacterial factor able to inhibit the cell-cycle of exposed cells, both of gastric and non-gastric origin. An integrated approach was adopted to isolate and characterise the molecule from the bacterial culture filtrate produced in a protein-free medium: size-exclusion chromatography, non-reducing gel electrophoresis, mass spectrometry, mutant analysis, recombinant protein expression and enzymatic assays. L-asparaginase was identified as the factor responsible for cell-cycle inhibition of fibroblasts and gastric cell lines. Its effect on cell-cycle was confirmed by inhibitors, a knockout strain and the action of recombinant L-asparaginase on cell lines. Interference with cell-cycle in vitro depended on cell genotype and was related to the expression levels of the concurrent enzyme asparagine synthetase. Bacterial subcellular distribution of L-asparaginase was also analysed along with its immunogenicity. H. pylori L-asparaginase is a novel antigen that functions as a cell-cycle inhibitor of fibroblasts and gastric cell lines. We give evidence supporting a role in the pathogenesis of H. pylori-related diseases and discuss its potential diagnostic application.

Helicobacter pyloril-asparaginase: a promising chemotherapeutic agent.

Bacterial L-asparaginases are amidohydrolases that catalyse the conversion of L-asparagine to L-aspartate and ammonia and are used as anti-cancer drugs. The current members of this class of drugs have several toxic side effects mainly due to their associated glutaminase activity. In the present study, we report the molecular cloning, biochemical characterisation and in vitro cytotoxicity of a novel L-asparaginase from the pathogenic strain Helicobacter pylori CCUG 17874. The recombinant enzyme showed a strong preference for L-asparagine over L-glutamine and, in contrast to most L-asparaginases, it exhibited a sigmoidal behaviour towards L-glutamine. The enzyme preserved full activity after 2 h incubation at 45 degrees C. In vitro cytotoxicity assays revealed that different cell lines displayed a variable sensitivity towards the enzyme, AGS and MKN28 gastric epithelial cells being the most affected. These findings may be relevant both for the interpretation of the mechanisms underlying H. pylori associated diseases and for biomedical applications.

Expression, purification and preliminary crystallographic studies on the catalytic region of the nonreceptor tyrosine kinase Fes.

The proto-oncogene tyrosine protein kinase c-fps/fes encodes a structurally unique protein (Fes) of the nonreceptor protein-tyrosine kinase (PTK) family. Its expression has been demonstrated in myeloid haematopoietic cells, vascular endothelial cells and in neurons. In human-derived and murine-derived cell lines, the activated form of this kinase can induce cellular transformation; moreover, it has been shown that Fes is involved in the regulation of cell-cell and cell-matrix interactions mediated by adherens junctions and focal adhesions. The N-terminus of Fes contains the FCH (Fps/Fes/Fer/CIP4 homology) domain, which is unique to the Fes/Fer kinase family. It is followed by three coiled-coil domains and an SH2 (Src-homology 2) domain. The catalytic region (Fes-CR) is located at the C-terminus of the protein. The successful expression, purification and crystallization of the catalytic part of Fes (Fes-CR) are described.

Comparative analysis of the neuronal activation and cardiovascular effects of nitroglycerin, sodium nitroprusside and L-arginine.

In this study, we compare the biological effects, Fos expression and cardiovascular responses induced in the rat, of different nitric oxide modulators (nitroglycerin, sodium nitroprusside and L-arginine). Nitroglycerin and sodium nitroprusside induced a similar pattern of neuronal activation in several areas, which include the paraventricular and supraoptic nuclei of the hypothalamus, central nucleus of the amygdala, parabrachial nucleus, locus coeruleus, ventrolateral medulla and nucleus tractus solitarius. However, only nitroglycerin activated the periaqueductal grey and nucleus trigeminalis caudalis. L-arginine-induced neuronal activation was restricted to the paraventricular and supraoptic nuclei of the hypothalamus. As regards cardiovascular effect, both nitroglycerin and sodium nitroprusside induced moderate hypotension (nitroglycerin: -23.3%, sodium nitroprusside: -24.3%) that lasted 40 min in the case of sodium nitroprusside and 80 min in the case of nitroglycerin. L-arginine did not significantly influence blood pressure. These data suggest that nitroglycerin, sodium nitroprusside and L-arginine are associated with different biological effects on both the central nervous system and the cardiovascular system. Of the NO-related drugs tested in this study, only nitroglycerin confirmed its ability to activate brainstem areas implicated in nociception.

Activation of the transcription factor NF-kappaB in the nucleus trigeminalis caudalis in an animal model of migraine.

The infusion of nitroglycerin (NTG) induces an inflammatory state in perivascular meningeal tissues of rat via the activation, inter alia, of nuclear factor kappa B (NF-kappaB). This phenomenon has been related to the mechanisms involved in the pathophysiology of migraine, a common neurovascular disorder. In the present study, we sought to elucidate whether NF-kappaB activation might have a role in the determinism of migraine attacks also at the neuronal level. Therefore, we investigated the transcriptional activity of NF-kappaB in the brainstem of rats systemically injected with NTG and killed 4h later. Activation of NF-kappaB in brain areas was detected by means of both the immunohistochemical technique and the Western blot analysis. A significant increase of nuclear immunostaining of p65, an indicator of NF-kappaB activation, was detected in lamina I and II of nucleus trigeminalis caudalis in rats injected with NTG when compared with the control group. Western blot analysis confirmed the activation of the NF-kappaB pathway showing an increase in the optical density of p65 in nuclear extracts of lower brainstem of rats injected with the nitric oxide (NO) donor. The present study contributes to expand on our understanding of the complex mechanisms by which NTG may trigger migraine-like headaches in migraineurs. Furthermore, these findings pave the way to new bio-molecular and pharmacological avenues for the development of innovative migraine therapies.

Risk stratification in the long-QT syndrome.

BACKGROUND: Mutations in potassium-channel genes KCNQ1 (LQT1 locus) and KCNH2 (LQT2 locus) and the sodium-channel gene SCN5A (LQT3 locus) are the most common causes of the long-QT syndrome. We stratified risk according to the genotype, in conjunction with other clinical variables such as sex and the length of the QT interval. METHODS: We evaluated 647 patients (386 with a mutation at the LQT1 locus, 206 with a mutation at the LQT2 locus, and 55 with a mutation at the LQT3 locus) from 193 consecutively genotyped families with the long-QT syndrome. The cumulative probability of a first cardiac event, defined as the occurrence of syncope, cardiac arrest, or sudden death before the age of 40 years and before the initiation of therapy, was determined according to genotype, sex, and the QT interval corrected for heart rate (QTc). Within each genotype we also assessed risk in the four categories derived from the combination of sex and QTc (<500 msec or > or =500 msec). RESULTS: The incidence of a first cardiac event before the age of 40 years and before the initiation of therapy was lower among patients with a mutation at the LQT1 locus (30 percent) than among those with a mutation at the LQT2 locus (46 percent) or those with a mutation at the LQT3 locus (42 percent) (P<0.001 by Fisher's exact test). Multivariate analysis showed that the genetic locus and the QTc, but not sex, were independent predictors of risk. The QTc was an independent predictor of risk among patients with a mutation at the LQT1 locus and those with a mutation at the LQT2 locus but not among those with a mutation at the LQT3 locus, whereas sex was an independent predictor of events only among those with a mutation at the LQT3 locus. CONCLUSIONS: The locus of the causative mutation affects the clinical course of the long-QT syndrome and modulates the effects of the QTc and sex on clinical manifestations. We propose an approach to risk stratification based on these variables.