Growth dynamics after historic disturbance in a montane forest and its implications for an endangered epiphytic lichen

Bolli J.C., Wagner H.H., Kalwij J.M., Werth S., Cherubini P., Scheidegger C. and Rigling A. 2008. Growth dynamics after historic disturbance in a montane forest and its implications for an endangered epiphytic lichen. Bot. Helv. 118: 111 – 127. Endangered forest species are often negatively affected by disturbances, which may have long-lasting effects on the distribution, abundance and genetic diversity of such species. To understand the effects of historic disturbances, detailed knowledge of the conditions for survival and recolonisation is needed, and this requires precise information on the perimeter and severity of historic disturbance events. We reconstructed a major historic disturbance (intensive logging followed by windthrow and fire in 1871) in the Swiss Jura mountains to analyse its effect on the disturbance-sensitive epiphytic lichen Lobaria pulmonaria. Tree-ring analysis of old and young Norway spruce trees (Picea abies L.), sampled systematically on a 100 m grid, revealed that (1) the disturbance was of intermediate severity, (2) a large, well-defined area of disturbance was created, and (3) an undisturbed zone remained in the centre of the disturbed area. Acomparison with lichen genetic data from a previous survey revealed that genetic diversity was particularly high in the remnant zone. These results suggest that the lichen survived there, and that it re-colonised the disturbed area both from the edge and from the remnant undisturbed zone. This illustrates that a detailed reconstruction of historic disturbances, as achieved with dendroecology, is very important for understanding the recolonisation process and thus, the conditions for the long-term persistence of disturbance-sensitive species in a dynamic landscape. Submitted 1 November 2007; Accepted 30 August 2008 Subject editor: Sabine Güsewell     

Systematic identification of novel cancer genes through analysis of deep shRNA perturbation screens

Systematic perturbation screens provide comprehensive resources for the elucidation of cancer driver genes. The perturbation of many genes in relatively few cell lines in such functional screens necessitates the development of specialized computational tools with sufficient statistical power. Here we developed APSiC (Analysis of Perturbation Screens for identifying novel Cancer genes) to identify genetic drivers and effectors in perturbation screens even with few samples. Applying APSiC to the shRNA screen Project DRIVE, APSiC identified well-known and novel putative mutational and amplified cancer genes across all cancer types and in specific cancer types. Additionally, APSiC discovered tumor-promoting and tumor-suppressive effectors, respectively, for individual cancer types, including genes involved in cell cycle control, Wnt/β-catenin and hippo signalling pathways. We functionally demonstrated that LRRC4B, a putative novel tumor-suppressive effector, suppresses proliferation by delaying cell cycle and modulates apoptosis in breast cancer. We demonstrate APSiC is a robust statistical framework for discovery of novel cancer genes through analysis of large-scale perturbation screens. The analysis of DRIVE using APSiC is provided as a web portal and represents a valuable resource for the discovery of novel cancer genes.     

Prostacyclin attenuates oxidative damage of myocytes by opening mitochondrial ATP-sensitive K+ channels via the EP3 receptor

Prostacyclin (PGI2) and the PGE family alleviate myocardial ischemia-reperfusion injury and limit oxidative damage. The cardioprotective effects of PGI2 have been traditionally ascribed to activation of IP receptors. Recent advances in prostanoid research have revealed that PGI2 can bind not only to IP, but also to EP, receptors, suggesting cross talk between PGI2 and PGEs. The mechanism(s) whereby PGI2 protects myocytes from oxidative damage and the specific receptors involved remain unknown. Thus fresh isolated adult rat myocytes were exposed to 200 microM H2O2 with or without carbaprostacyclin (cPGI2), IP-selective agonists, and ONO-AE-248 (an EP3-selective agonist). Cell viability was assessed by trypan blue exclusion after 30 min of H2O2 superfusion. cPGI2 and ONO-AE-248 significantly improved cell survival during H2O2 superfusion; IP-selective agonists did not. The protective effect of cPGI2 and ONO-AE-248 was completely abrogated by pretreatment with 5-hydroxydecanoate or glibenclamide. In the second series of experiments, the mitochondrial ATP-sensitive K+ (K(ATP)) channel opener diazoxide (Dx) reversibly oxidized flavoproteins in control myocytes. Exposure to prostanoid analogs alone had no effect on flavoprotein fluorescence. A second application of Dx in the presence of cPGI2 or ONO-AE-248 significantly increased flavoprotein fluorescence compared with Dx alone, but IP-selective agonists did not. This study demonstrates that PGI2 analogs protect cardiac myocytes from oxidative stress mainly via activation of EP3. The data also indicate that activation of EP3 receptors primes the opening of mitochondrial K(ATP) channels and that this mechanism is essential for EP3-dependent protection.     

Nitric oxide (NO) induces nitration of protein kinase Cepsilon (PKCepsilon ), facilitating PKCepsilon translocation via enhanced PKCepsilon -RACK2 interactions: a novel mechanism of no-triggered activation of PKCepsilon

Activation of protein kinase C (PKC) epsilon by nitric oxide (NO) has been implicated in the development of cardioprotection. However, the cellular mechanisms underlying the activation of PKCepsilon by NO remain largely unknown. Nitration of protein tyrosine residues has been shown to alter functions of a variety of proteins, and NO-derived peroxynitrite is known as a strong nitrating agent. In this investigation, we demonstrate that NO donors promote translocation and activation of PKCepsilon in an NO- and peroxynitrite-dependent fashion. NO induces peroxynitrite-mediated tyrosine nitration of PKCepsilon in rabbit cardiomyocytes in vitro, and nitrotyrosine residues were also detected on PKCepsilon in vivo in the rabbit myocardium preconditioned with NO donors. Furthermore, coimmunoprecipitation of PKCepsilon and its receptor for activated C kinase, RACK2, illustrated a peroxynitrite-dependent increase in PKCepsilon-RACK2 interactions in NO donor-treated cardiomyocytes. Moreover, using an enzyme-linked immunosorbent assay-based protein-protein interaction assay, PKCepsilon proteins treated with the peroxynitrite donor SIN-1 exhibited enhanced binding to RACK2 in an acellular environment. Our data demonstrate that post-translational modification of PKCepsilon by NO donors, namely nitration of PKCepsilon, facilitates its interaction with RACK2 and promotes translocation and activation of PKCepsilon. These findings offer a plausible novel mechanism by which NO activates the PKC signaling pathway.     

Protein tyrosine kinase signaling is necessary for NO donor-induced late preconditioning against myocardial stunning

Although protein tyrosine kinases (PTKs) signaling has been implicated in the late phase of ischemic preconditioning (PC), it is unknown whether PTK signaling is necessary for the development of nitric oxide (NO) donor-induced late PC. Thus conscious rabbits underwent a sequence of six 4-min coronary occlusion (O)/4-min reperfusion (R) cycles followed by a 5-h recovery period of reperfusion for 3 consecutive days (days 1, 2, and 3). On day 0 (24 h before the 6 O/R cycles on day 1), rabbits received no treatment (control), the NO donor diethylenetriamine (DETA)/NO (DETA/NO), the PTK inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), or DETA/NO plus PP2 (DETA/NO + PP2). In control rabbits (n = 6), the six O/R cycles on day 1 resulted in delayed functional recovery, indicating severe myocardial stunning. In rabbits pretreated with DETA/NO (n = 5) on day 1, myocardial stunning caused by the six O/R cycles on day 1 was markedly attenuated, with a significant reduction ( approximately 60%) in the total deficit of wall thickening (WTh) compared with controls, indicating that DETA/NO induced a late PC effect against stunning. However, in rabbits pretreated with DETA/NO + PP2 (n = 5), the total deficit of WTh was significantly greater than that in rabbits treated with DETA/NO alone and was similar to that in controls, indicating that PP2 prevented the development of DETA/NO-induced late PC. In rabbits pretreated with PP2 on day 0 (n = 4), the total deficit of WTh was similar to that in controls, indicating that PP2 does not affect myocardial stunning in itself. We conclude that a PTK-dependent signaling mechanism is necessary for the development of NO donor-induced late PC against myocardial stunning in conscious rabbits.     

Nicorandil induces late preconditioning against myocardial infarction in conscious rabbits

Nicorandil has been shown to induce an infarct-limiting effect similar to that induced by the early phase of ischemic preconditioning (PC). The goals of this study were to determine whether nicorandil induces a delayed cardioprotection that is analogous to the late phase of ischemic PC and, if so, whether nicorandil-induced late PC is associated with upregulation of cardioprotective proteins. Chronically instrumented, conscious rabbits received vehicle (intravenous normal saline; control group, n = 10), nicorandil (100 microg/kg bolus + 30 microg x kg(-1) x min(-1) i.v. for 60 min; nicorandil group, n = 10), or ischemic PC (6 cycles of 4-min coronary occlusion/4-min reperfusion; PC group, n = 8). Twenty-four hours later, rabbits underwent a 30-min coronary occlusion, followed by 3 days of reperfusion. Myocardial infarct size was significantly reduced in rabbits pretreated with nicorandil (27.5 +/- 5.3% of the risk region) or with ischemia (30.3 +/- 4.2%) versus controls (59.1 +/- 4.7%, P < 0.05 vs. both). Furthermore, the expression of cyclooxygenase-2 (COX-2) and Bcl-2 was significantly elevated (+38% and +126%, respectively; P < 0.05) in myocardium of rabbits given nicorandil 24 h earlier versus controls. We conclude that nicorandil induces delayed cardioprotection against myocardial infarction similar to that afforded by the late phase of ischemic PC, possibly by upregulating COX-2 and Bcl-2.     

PKCepsilon activation induces dichotomous cardiac phenotypes and modulates PKCepsilon-RACK interactions and RACK expression

Receptors for activated C kinase (RACKs) have been shown to facilitate activation of protein kinase C (PKC). However, it is unknown whether PKC activation modulates RACK protein expression and PKC-RACK interactions. This issue was studied in two PKCepsilon transgenic lines exhibiting dichotomous cardiac phenotypes: one exhibits increased resistance to myocardial ischemia (cardioprotected phenotype) induced by a modest increase in PKCepsilon activity (228 +/- 23% of control), whereas the other exhibits cardiac hypertrophy and failure (hypertrophied phenotype) induced by a marked increase in PKCepsilon activity (452 +/- 28% of control). Our data demonstrate that activation of PKC modulates the expression of RACK isotypes and PKC-RACK interactions in a PKCepsilon activity- and dosage-dependent fashion. We found that, in mice displaying the cardioprotected phenotype, activation of PKCepsilon enhanced RACK2 expression (178 +/- 13% of control) and particulate PKCepsilon-RACK2 protein-protein interactions (178 +/- 18% of control). In contrast, in mice displaying the hypertrophied phenotype, there was not only an increase in RACK2 expression (330 +/- 33% of control) and particulate PKCepsilon-RACK2 interactions (154 +/- 14% of control) but also in RACK1 protein expression (174 +/- 10% of control). Most notably, PKCepsilon-RACK1 interactions were identified in this line. With the use of transgenic mice expressing a dominant negative PKCepsilon, we found that the changes in RACK expression as well as the attending cardiac phenotypes were dependent on PKCepsilon activity. Our observations demonstrate that RACK expression is dynamically regulated by PKCepsilon and suggest that differential patterns of PKCepsilon-RACK interactions may be important determinants of PKCepsilon-dependent cardiac phenotypes.     

Differential role of K(ATP) channels in late preconditioning against myocardial stunning and infarction in rabbits

The role of ATP-sensitive potassium (K(ATP)) channels in the late phase of ischemic preconditioning (PC) remains unclear. Furthermore, it is unknown whether K(ATP) channels serve as end effectors both for late PC against infarction and against stunning. Thus, in phase I of this study, conscious rabbits underwent a 30-min coronary occlusion (O) followed by 72 h of reperfusion (R) with or without ischemic PC (6 4-min O/4-min R cycles) 24 h earlier. Late PC reduced infarct size approximately 46% versus controls. The K(ATP) channel blocker 5-hydroxydecanoic acid (5-HD), given 5 min before the 30-min O, abrogated the infarct-sparing effect of late PC but did not alter infarct size in non-PC rabbits. In phase II, rabbits underwent six 4-min O/4-min R cycles for 3 consecutive days (days 1, 2, and 3). In controls, the total deficit of systolic wall thickening (WTh) after the sixth reperfusion was reduced by 46% on day 2 and 54% on day 3 compared with day 1, indicating a late PC effect against myocardial stunning. Neither 5-HD nor glibenclamide, given on day 2, abrogated late PC. The K(ATP) channel opener diazoxide, given on day 1, attenuated stunning, and this effect was completely blocked by 5-HD. Thus the same dose of 5-HD that blocked the antistunning effect of diazoxide failed to block the antistunning effects of late PC. Furthermore, when diazoxide was administered in PC rabbits on day 2, myocardial stunning was further attenuated, indicating that diazoxide and late PC have additive anti-stunning effects. We conclude that K(ATP) channels play an essential role in late PC against infarction but not in late PC against stunning, revealing an important pathogenetic difference between these two forms of cardioprotection.     

l-Proline reduces IgG dimer content and enhances the stability of intravenous immunoglobulin (IVIG) solutions

Liquid intravenous immunoglobulin (IVIG) products offer improved convenience in preparation but often lack sufficient stability to allow room temperature storage. Furthermore, clinical tolerability may be affected due to formation of idiotype/anti-idiotype IgG dimers and/or aggregates. Here we report on the development of a 10% IVIG formulation with optimized stability achieved by the use of l-proline. The stability of concentrated liquid IVIG was strongly pH dependent. Aggregate formation, yellowish discoloration of the solution and loss of anti-hepatitis B surface antigen (HBs) antibody activity was minimal at intermediate pH (pH 4.8–5.3). Fragmentation of IgG was highest at low pH (pH 4.1). Idiotype/anti-idiotype IgG dimer formation was highest at neutral pH and was reduced with decreasing pH. The presence of l-proline further improved stability by inhibiting protein aggregation, reducing loss of anti-HBs antibody activity and decreasing coloring, particularly compared with glycine formulations. The IgG dimer content was up to 30% lower in solutions containing l-proline compared with those containing glycine or other stabilizers. In conclusion, a weakly acidic pH of approximately 5 and l-proline as stabilizer are optimal conditions for long-term stability of a liquid IVIG. l-proline, an amphiphilic, naturally occurring amino acid, is superior to glycine in restricting IgG dimer formation.     

Fatigue effects on the coordinative pattern during cycling: Kinetics and kinematics evaluation

The aim of the present study was to analyze the net joint moment distribution, joint forces and kinematics during cycling to exhaustion. Right pedal forces and lower limb kinematics of ten cyclists were measured throughout a fatigue cycling test at 100% of PO_MAX. The absolute net joint moments, resultant force and kinematics were calculated for the hip, knee and ankle joint through inverse dynamics. The contribution of each joint to the total net joint moments was computed. Decreased pedaling cadence was observed followed by a decreased ankle moment contribution to the total joint moments in the end of the test. The total absolute joint moment, and the hip and knee moments has also increased with fatigue. Resultant force was increased, while kinematics has changed in the end of the test for hip, knee and ankle joints. Reduced ankle contribution to the total absolute joint moment combined with higher ankle force and changes in kinematics has indicated a different mechanical function for this joint. Kinetics and kinematics changes observed at hip and knee joint was expected due to their function as power sources. Kinematics changes would be explained as an attempt to overcome decreased contractile properties of muscles during fatigue.