Effect of remote ischaemic conditioning on clinical outcomes in patients with acute myocardial infarction (CONDI-2/ERIC-PPCI): a single-blind randomised controlled trial.

BACKGROUND: Remote ischaemic conditioning with transient ischaemia and reperfusion applied to the arm has been shown to reduce myocardial infarct size in patients with ST-elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention (PPCI). We investigated whether remote ischaemic conditioning could reduce the incidence of cardiac death and hospitalisation for heart failure at 12 months. METHODS: We did an international investigator-initiated, prospective, single-blind, randomised controlled trial (CONDI-2/ERIC-PPCI) at 33 centres across the UK, Denmark, Spain, and Serbia. Patients (age >18 years) with suspected STEMI and who were eligible for PPCI were randomly allocated (1:1, stratified by centre with a permuted block method) to receive standard treatment (including a sham simulated remote ischaemic conditioning intervention at UK sites only) or remote ischaemic conditioning treatment (intermittent ischaemia and reperfusion applied to the arm through four cycles of 5-min inflation and 5-min deflation of an automated cuff device) before PPCI. Investigators responsible for data collection and outcome assessment were masked to treatment allocation. The primary combined endpoint was cardiac death or hospitalisation for heart failure at 12 months in the intention-to-treat population. This trial is registered with ClinicalTrials.gov (NCT02342522) and is completed. FINDINGS: Between Nov 6, 2013, and March 31, 2018, 5401 patients were randomly allocated to either the control group (n=2701) or the remote ischaemic conditioning group (n=2700). After exclusion of patients upon hospital arrival or loss to follow-up, 2569 patients in the control group and 2546 in the intervention group were included in the intention-to-treat analysis. At 12 months post-PPCI, the Kaplan-Meier-estimated frequencies of cardiac death or hospitalisation for heart failure (the primary endpoint) were 220 (8·6%) patients in the control group and 239 (9·4%) in the remote ischaemic conditioning group (hazard ratio 1·10 [95% CI 0·91-1·32], p=0·32 for intervention versus control). No important unexpected adverse events or side effects of remote ischaemic conditioning were observed. INTERPRETATION: Remote ischaemic conditioning does not improve clinical outcomes (cardiac death or hospitalisation for heart failure) at 12 months in patients with STEMI undergoing PPCI. FUNDING: British Heart Foundation, University College London Hospitals/University College London Biomedical Research Centre, Danish Innovation Foundation, Novo Nordisk Foundation, TrygFonden

Evolution of polysaccharide hydrolase substrate-specificity: catalytic amino-acids are conserved in barley 1,3-1,4-glucanase and 1,3-beta-glucanase

Catalytic amino acid residues in a 1,3-beta-D-glucan 3-glucanohydrolase (EC 3.2.1.39) and a homologous 1,3-1,4-beta-D-glucan 4-glucanohydrolase (EC 3.2.1.73) from barley have been investigated. To identify amino acids responsible for protonation of the glycosidic oxygen during hydrolysis, carbodiimide-mediated labeling of the enzymes with [14C]glycine ethyl ester was performed. This resulted in loss of activity and specific modification of the Glu288 residues in both enzymes. The stoichiometry of labeling was approximately 1:1, and modification was reduced in the presence of substrate analogues. Based on these data, the Glu288 residues are likely to be present at the active sites of the respective enzymes and may represent the catalytic acids in the hydrolytic reaction. The catalytic nucleophiles of the two enzymes were investigated by labeling with specific, mechanism-based epoxyalkyl-beta-oligoglucosides. Amino acid residues Glu232 and Glu231 were identified as the likely catalytic nucleophiles in the 1,3-1,4- and 1,3-beta-glucanases, respectively. Thus the position of the catalytic nucleophile and the putative proton donating amino acids in the two classes of beta-glucan endohydrolases are conserved. The acquisition of distinct substrate specificities in the evolution of these related enzymes may therefore not require the recruitment of novel catalytic amino acids but rather differences in their positioning at the active site and/or changes in substrate binding residues

Isolation of a cDNA clone specifying rat chaperonin-10, a stress-inducible mitochondrial matrix protein synthesized without a cleavable presequence

We have isolated a cDNA clone encoding chaperonin 10 from rat liver. The cDNA specifies a protein of 102 amino acids which, when transcribed and translated in vitro, yields a single basic product (pI > 9) that co-migrates exactly with the heat shock inducible cpn10 of rat hepatoma cells during 2D gel-electrophoresis. It is concluded that cpn10, unlike the majority of nuclear-encoded proteins of the mitochondrial matrix, is synthesised without a cleavable targeting signal and that, following removal of the initiating methionine, it becomes acetylated prior to mitochondrial import. Incubation of 3H- or 35S-labelled cpn10 with mitochondria confirms these conclusions and shows that cpn10 is imported into mitochondria in an energy-dependent process which is inhibited by the presence of 2,4-dinitrophenol

Differences in active-site structure in a family of beta-glucan endohydrolases deduced from the kinetics of inactivation by epoxyalkyl beta-oligoglucosides

The active sites of a spectrum of beta-glucan endohydrolases with distinct, but related substrate specificities have been probed using a series of epoxyalkyl beta-glycosides of glucose, cellobiose, cellotriose, laminaribiose, laminaritriose, 3O-beta-D-glucosyl-cellobiose and 4O-beta-D-glucosyl-laminaribiose with different aglycon chain lengths. The inactivation of each of the endohydrolases by these compounds results from active site-directed inhibitor action, as indicated by the dependence of the inactivation rate on pH, glycosyl chain length and linkage position, aglycon length, and the protective effect of disaccharides derived from the natural substrates. Comparisons of inhibitor specificity between a Bacillus subtilis 1,3;1,4-beta-D-glucan 4-glucanohydrolase (EC 3.2.1.73), a Streptomyces cellulase (EC 3.2.1.4), a Schizophyllum commune cellulase (EC 3.2.1.4), a Rhizopus arrhizus 1,3-(1,3;1,4)-beta-D-glucan 3(4)-glucanohydrolase (EC 3.2.1.6), and a Nicotiana glutinosa 1,3-beta-D-glucan 3-glucanohydrolase (EC 3.2.1.39) demonstrated different tolerances for glycosyl linkage positions in the inactivation process and a critical role of aglycon length reflecting differences in the active site geometry of the enzymes. For the B. subtilis endohydrolase it was concluded that the aglycon residue of the inhibitor spans the glycosyl binding subsite occupied by the 3-substituted glucosyl residue involved in the glucosidic linkage cleaved in the natural substrate. Appropriate positioning of the inhibitor epoxide group with respect to the catalytic amino acids in the active site is crucial to the inactivation step and the number of glucosyl residues in the inhibitor affects aglycon chain length specificity. The importance of this effect differs between the glucanases tested and may be related to the number of glycosyl binding subsites in the active site

Posttranslational processing of barley beta-glucan endohydrolases in the baculovirus insect cell expression system

Two cDNAs encoding barley (1→3,1→4)-β-glucanase (EC 3.2.1.73) isoenzymes EI and EII have been expressed in Spodoptera frugiperda (Sf9) cell cultures using the baculovirus AcNPV vector. Modifications to both the 5′ and 3′ ends of the cDNAs were required before satisfactory levels of expression were obtained. The modified cDNAs directed high levels of (1→3,1→4)-β-glucanase expression in the Sf9 insect cell cultures, with yields of 10 mg/liter of isoenzyme EI (expEI) and 15 mg/liter of isoenzyme EII (expEII). Amino acid sequence analyses showed that the expressed enzymes were processed correctly at their amino termini. However, affinity chromatography of the isoenzyme expEII on concanavalin-A (conA)-Sepharose indicated that, although the enzyme is glycosylated, the structures of the carbohydrate chains differ from those of the native enzyme. When a cDNA encoding the homologous barley (1→3)-β-glucanase (EC 3.2.1.39) isoenzyme GII was expressed in insect cells, aberrant amino-terminal processing of the nascent polypeptide was sometimes observed. The forms with incompletely removed signal peptides retained their substrate specificity, but exhibited slightly reduced catalytic efficiency, altered Chromatographic behavior, and reduced stability at elevated temperatures. The results show that high levels of expression of recombinant plant proteins can be obtained in insect cells, but they emphasize the need to characterize thoroughly the products that are expressed in the heterologous insect cell system before comparisons are made with the native enzyme or with engineered enzyme mutants