Richard A. Knight

S -nitrosylation regulates apoptosis

Nitric oxide (NO) modulates the biological activity of proteins by direct interactions with their iron centres. It can also S-nitrosylate cysteines to form S-nitrosothiols. Such reactions affect the activity of membrane-bound, cytosolic and nuclear proteins including the NMDA receptor, haemoglobin and transcription factors such as NF-κB and OxyR. NO is potentially toxic, inducing both apoptosis and necrosis. Here we show that NO-mediated S-nitrosylation of the cysteine-containing enzymes that mediate apoptosis (caspases and tissue-transglutaminase, tTG) regulates the balance between apoptosis and necrosis.

p73 Induces Apoptosis via PUMA Transactivation and Bax Mitochondrial Translocation

p73, an important developmental gene, shares a high sequence homology with p53 and induces both G1 cell cycle arrest and apoptosis. However, the molecular mechanisms through which p73 induces apoptosis are unclear. We found that p73-induced apoptosis is mediated by PUMA (p53 up-regulated modulator of apoptosis) induction, which, in turn, causes Bax mitochondrial translocation and cytochrome c release. Overexpression of p73 isoforms promotes cell death and bax promoter transactivation in a time-dependent manner. However, the kinetics of apoptosis do not correlate with the increase of Bax protein levels. Instead, p73-induced mitochondrial translocation of Bax is kinetically compatible with the induction of cell death. p73 is localized in the nucleus and remains nuclear during the induction of cell death, indicating that the effect of p73 on Bax translocation is indirect. The ability of p73 to directly transactivate PUMA and the direct effect of PUMA on Bax conformation and mitochondrial relocalization suggest a molecular link between p73 and the mitochondrial apoptotic pathway. Our data therefore indicate that PUMA-mediated Bax mitochondrial translocation, rather than its direct transactivation, correlates with cell death. Finally, human ΔNp73, an isoform lacking the amino-terminal transactivation domain, inhibits TAp73-induced as well as p53-induced apoptosis. The ΔNp73 isoforms seem therefore to act as dominant negatives, repressing the PUMA/Bax system and, thus, finely tuning p73-induced apoptosis. Our findings demonstrate that p73 elicits apoptosis via the mitochondrial pathway using PUMA and Bax as mediators.

Different Signaling Pathways Induce Apoptosis in Endothelial Cells and Cardiac Myocytes During Ischemia/Reperfusion Injury

Apoptosis contributes, with necrosis, to the cardiac cell loss after ischemia/reperfusion injury. The apoptotic cascade is initiated either by mitochondrial damage and activation of caspase-9 or by death receptor ligation and activation of caspase-8. In the present study, performed in the isolated rat heart exposed either to ischemia alone or ischemia followed by reperfusion, cleavage of caspase-9 was observed primarily in endothelial cells. Conversely, caspase-8 cleavage was only found in cardiomyocytes, where it progressively increased throughout reperfusion. Addition of a specific caspase-9 inhibitor to the perfusate before ischemia prevented endothelial apoptosis, whereas preischemic infusion of a specific caspase-8 inhibitor affected only myocyte apoptosis. Additionally, caspase-8–mediated BID processing was observed only during reperfusion. Production of tBID then sustains mitochondrial injury and perpetuates caspase-9 activation.

CRH-like peptides protect cardiac myocytes from lethal ischaemic injury.

Simulated ischaemia causes both necrotic and apoptotic death of primary cultures of neonatal rat cardiac myocytes. Simulated ischaemia is associated with increased expression of urocortin mRNA and with the release of urocortin peptide into the medium. Exogenous urocortin is more potent than corticotropin releasing hormone (CRH) in protecting cardiac myocytes from necrotic and apoptotic death induced by ischaemia, and the cardioprotective effects of ischaemia-preconditioned media are abrogated by antagonists to the CRH family of peptides. Simulated ischaemia increases cardiac myocyte expression of CCAAT enhancer binding (C/EBP) transcription factors, and of the p65 subunit of NFkappaB, and reporter activity of a construct incorporating a fragment of the urocortin promoter containing a C/EBP consensus site is also enhanced by simulated ischaemia. The data suggest that ischaemia, acting partly through increased expression of C/EBP transactivators, increases expression of urocortin mRNA, which is rapidly translated to the mature form. The mature peptide is rapidly released, and exerts autocrine/paracrine protective effects through the cardiac CRH-R2 receptor which preferentially binds urocortin.

urocortin promotes hemodynamic and bioenergetic recovery and improves cell survival in the isolated rat heart exposed to ischemia/reperfusion

OBJECTIVES This study evaluates the hemodynamic, bioenergetic and cytoprotective effects of urocortin (Ucn) in the isolated rat heart exposed to ischemia (I)/reperfusion (R). BACKGROUND We have previously demonstrated that administration of exogenous Ucn reduces infarct size in ischemic-reperfused rat hearts. METHODS Urocortin 10(-8)M was added to the perfusate before I, before I and during R, and during R alone in the isolated pulsed rat heart exposed to 35 min I followed by 60 min R. RESULTS Partial to complete recovery of diastolic pressure and developed pressure was seen irrespective of when Ucn was perfused. In particular, beneficial effects are observed when Ucn is only given during R. Urocortin given only before I, and before I and over R, although not during R alone, also produces significant recovery of high-energy phosphate pools. In each group, improvement in ventricular function is associated with reduction both in myocardial damage, assessed by creatine phosphokinase release, and in endothelial cell and cardiomyocyte apoptosis, assessed by caspase 3 activity and fluorescent-based terminal deoxynucleotidyl transferase mediated nick end labelling enhanced with counterstains. These improvements in ventricular performance, bioenergetics and cell survival are not secondary to any inotropic effects of Ucn. CONCLUSIONS This is the first report to show enhanced cardiac function induced by Ucn during I/R. Because the cytoprotective and functional benefits are still produced when Ucn is given only at R, these data suggest that Ucn may be useful clinically in the management of myocardial infarction.

Human Rhinovirus Selectively Modulates Membranous and Soluble Forms of Its Intercellular Adhesion Molecule–1 (ICAM-1) Receptor to Promote Epithelial Cell Infectivity

Human rhinoviruses are responsible for many upper respiratory tract infections. 90% of rhinoviruses utilize intercellular adhesion molecule-1 (ICAM-1) as their cellular receptor, which also plays a critical role in recruitment of immune effector cells. Two forms of this receptor exist; membrane-bound (mICAM-1) and soluble ICAM-1 (sICAM-1). The soluble receptor may be produced independently from the membrane-bound form or it may be the product of proteolytic cleavage of mICAM-1. The ratio of airway epithelial cell expression of mICAM-1 to the sICAM-1 form may influence cell infectivity and outcome of rhinovirus infection. We therefore investigated the effect of rhinovirus on expression of both ICAM-1 receptors in normal human bronchial epithelial cells. We observed separate distinct messenger RNA transcripts coding for mICAM-1 and sICAM-1 in these cells, which were modulated by virus. Rhinovirus induced mICAM-1 expression on epithelial cells while simultaneously down-regulating sICAM-1 release, with consequent increase in target cell infectivity. The role of protein tyrosine kinases was investigated as a potential mechanistic pathway. Rhinovirus infection induced rapid phosphorylation of intracellular tyrosine kinase, which may be critical in up-regulation of mICAM-1. Elucidation of the underlying molecular mechanisms involved in differential modulation of both ICAM-1 receptors may lead to novel therapeutic strategies.

Th2 cytokines exert a dominant influence on epithelial cell expression of the major group human rhinovirus receptor, ICAM-1

Intercellular adhesion molecule (ICAM)-1 is a cell receptor important in both human rhinovirus (HRV) attachment and immune effector cell mobilization. The level of expression of ICAM-1 by epithelial cells (EC) therefore plays a crucial role in the intricate biological phenomena underlying viral binding, host infection and consequent inflammatory events. As T-helper (Th)2 lymphocytes predominate within the asthmatic airway, the influence was evaluated of Th2-associated mediators in the modulation of ICAM-1 expression on uninfected and HRV-infected EC. H292 EC were cultured in vitro, with varying concentrations of interleukin (IL)-4, IL-5, IL-10 and IL-13 for 24 h and then infected with live HRV-14. Surface ICAM-1 expression was assessed by immunocytochemistry. Infection with HRV-14 resulted in a twofold increase in ICAM-1 expression. IL-4, IL-5, IL-10 and IL-13 produced a 2.7-5.1-fold enhancement of ICAM-1 expression of uninfected cells and caused approximately a further twofold increase in infected cells over the expression induced by HRV infection itself. Interferon-gamma in combination with each Th2-associated cytokine only slightly reduced, but did not override, the Th2-induced level of ICAM-1 expression on both uninfected and virus-infected EC. These data suggest that the effects of Th2-associated cytokines on intercellular adhesion molecule-1 expression and recovery of infectious virus are dominant over the effects of the Th1-associated cytokines such as interferon-gamma. Since the airway mucosa in atopic asthma is predominantly infiltrated by Th2 lymphocytes, these results could explain both the increased susceptibility to human rhinovirus infection in asthmatic patients and the associated exacerbation of asthma symptoms.

Retinoids and the control of growth/death decisions in human neuroblastoma cell lines

Cell proliferation, the balance between mitosis and apoptosis is the result of the continuous integration of a number of different signal transduction pathways stimulated in a cell at any given point in its life. Neuroblastoma cells regulate the switch between mitosis and death, according both to intrinsic factors and extrinsic factors, such as growth factor withdrawal and action of the vitamin A derivative, retinoic acid. In this review, we describe the balance of some factors regulating growth and death of human neuroblastoma cells in vitro. These dynamic studies are necessarily performed on cell lines, which offer controlled conditions enabling the disection of the complex stimuli mediating survival and growth (IGF, trk, BDNF) and death (transglutaminase, free radicals, Bcl-2). Although the conclusions drawn may therefore not be directly applicable to tumour cells in vivo, the results herein discussed are of sufficient significance to warrant in vivo relevance.

Quantitative assessment of cardiac myocyte apoptosis in tissue sections using the fluorescence-based tunel technique enhanced with counterstains.

Apoptosis is a distinct form of cell death, induced, for example, by ischaemia/reperfusion injury, that results in characteristic alterations in cell morphology and fate. In tissue sections, the most commonly used technique to detect apoptosis is terminal deoxynucleotidyl transferase mediated nick end labelling (TUNEL) staining which labels the ends of DNA strand breaks characteristic of the apoptotic process. However, without the employment of additional staining, TUNEL is only a qualitative procedure that gives no information about the proportion of negative cells nor the cell type undergoing apoptosis. We have utilised propidium iodide (PI) as a counterstain to visualise TUNEL negative nuclei together with anti-desmin antibody in order to assess quantitatively apoptosis in specific cell types. The procedure has been evaluated in tissue sections from isolated perfused rat hearts subjected to ischaemia and reperfusion. Hearts were cross-sectioned into four 2.5 mm thick slices which were fixed in 4% formaldehyde and embedded in paraffin. Serial sections (5 microns) were cut, dewaxed and pretreated by incubation with trypsin at 37 degrees C for 30 min. After the employment of the TUNEL assay, sections were labelled with anti-desmin antibody, counterstained with PI and finally examined by confocal fluorescent microscopy. Apoptosis was not seen in sections from hearts subjected to ischaemia alone nor in control hearts. After 35 min of ischaemia the percentages of TUNEL positive cells were very low both in myocytes (0.1%) and in non-myocytes (0.3%). In ischaemic-reperfused hearts, the number of TUNEL positive cells was only significantly higher in vascular cells (44+/-5%) and cardiac myocytes (6+/-2%). This simple method therefore allows quantification of apoptosis in myocytic and non-myocytic cells in tissue sections. Use of alternative immunohistochemical markers would permit adaptation of the method to the quantitative assessment of apoptosis in other tissues.

Incidence and expression of the N1303K mutation of the cystic fibrosis (CFTR) gene.

SummaryThe N1303K mutation was identified in the second nucleotide binding fold of the cystic fibrosis (CF) gene last year. We have gathered data from laboratories throughout Europe and the United States of America in order to estimate its frequency and to attempt to characterise the clinical manifestations of this mutation. N1303K, identified on 216 of nearly 15000 CF chromosomes tested, accounts for 1.5% of all CF chromosomes. The frequency of the N1303K allele varies significantly between countries and ethnic groups, being more common in Southern than in Northern Europe. This variation is independent of the AF508 allele. It was not found on UK Asian, American Black or Australian chromosomes. N1303K is associated with four different linked marker haplotypes for the polymorphic markers XV-2c, KM.19 and pMP6d-9. Ten patients are homozygous for this mutation, whereas 106 of the remainder carry one of 12 known CF mutations in the other CF allele. We classify N1303K as a “severe” mutation with respect to the pancreas, but can find no correlation between this mutation, in either the homozygous or heterozygous state, and the severity of lung disease.