Cryo-EM of ribosomal 80S complexes with termination factors reveals the translocated Cricket Paralysis Virus IRES

The cricket paralysis virus (CrPV) uses an internal ribosomal entry site (IRES) to hijack the ribosome. In a remarkable RNA-based mechanism involving neither initiation factor nor initiator tRNA, the CrPV IRES jumpstarts translation in the elongation phase from the ribosomal A site. Here, we present cryoelectron microscopy (cryo-EM) maps of 80S⋅CrPV-STOP ⋅ eRF1 ⋅ eRF3 ⋅ GMPPNP and 80S⋅CrPV-STOP ⋅ eRF1 complexes, revealing a previously unseen binding state of the IRES and directly rationalizing that an eEF2-dependent translocation of the IRES is required to allow the first A-site occupation. During this unusual translocation event, the IRES undergoes a pronounced conformational change to a more stretched conformation. At the same time, our structural analysis provides information about the binding modes of eRF1 ⋅ eRF3 ⋅ GMPPNP and eRF1 in a minimal system. It shows that neither eRF3 nor ABCE1 are required for the active conformation of eRF1 at the intersection between eukaryotic termination and recycling

Anaesthetics and cardiac preconditioning. Part II. Clinical implications

There is compelling evidence that preconditioning occurs in humans. Experimental studies with potential clinical implications as well as clinical studies evaluating ischaemic, pharmacological and anaesthetic cardiac preconditioning in the perioperative setting are reviewed. These studies reveal promising results. However, there are conflicting reports on the efficacy of preconditioning in the diseased and aged myocardium. In addition, many anaesthetics and a significant number of perioperatively administered drugs affect the activity of cardiac sarcolemmal and mitochondrial KATP channels, the end‐effectors of cardiac preconditioning, and thereby markedly modulate preconditioning effects in myocardial tissue. Although these modulatory effects on KATP channels have been investigated almost exclusively in laboratory investigations, they may have potential implications in clinical medicine. Important questions regarding the clinical utility and applicability of perioperative cardiac preconditioning remain unresolved and need more experimental work and randomized controlled clinical trials. Br J Anaesth 2003; 91: 566-7

Effect of rofecoxib on platelet aggregation and blood loss in gynaecological and breast surgery compared with diclofenac

Background. Non‐selective cyclooxygenase (COX) inhibitors or non‐steroidal anti‐ inflammatory drugs (NSAIDs) are frequently omitted for perioperative pain relief because of potential side‐effects. COX‐2‐selective inhibitors may have a more favourable side‐effect profile. This study tested the hypothesis that the COX‐2‐selective inhibitor rofecoxib has less influence on platelet function than the NSAID diclofenac in gynaecological surgery. In addition, analgesic efficacy and side‐effects of the two drugs were compared. Methods. In this single‐centre, prospective, double‐blind, active controlled study, women undergoing vaginal hysterectomy (n=25) or breast surgery (n=25) under general anaesthesia received preoperatively 50 mg of rofecoxib p.o. followed 8 and 16 h later by two doses of placebo or three doses of diclofenac 50 mg p.o. at the same time points. We assessed arachidonic acid‐stimulated platelet aggregation before and 4 h after the first dose of study medication, estimated intraoperative blood loss, and haemoglobin loss until the first morning after surgery. Analgesic efficacy, use of rescue analgesics, and side‐effects were also recorded. Results. In the rofecoxib group, stimulated platelet aggregation was disturbed less (P=0.02), and estimated intraoperative blood loss (P=0.01) and the decrease in haemoglobin were lower (P=0.01). At similar pain ratings, the use of anti‐emetic drugs was less in the rofecoxib group (P=0.03). Conclusion. Besides having a smaller effect on platelet aggregation, one oral dose of rofecoxib 50 mg given before surgery provided postoperative analgesia similar to that given by three doses of diclofenac 50 mg and was associated with less use of anti‐emetics and less surgical blood loss in gynaecological surgery compared with diclofenac. Br J Anaesth 2004; 92: 523-3

Structure of the mammalian ribosome as it decodes the selenocysteine UGA codon

The elongation of eukaryotic selenoproteins relies on a poorly understood process of interpreting in-frame UGA stop codons as selenocysteine (Sec). We used cryo-electron microscopy to visualize Sec UGA recoding in mammals. A complex between the noncoding Sec-insertion sequence (SECIS), SECIS-binding protein 2 (SBP2), and 40S ribosomal subunit enables Sec-specific elongation factor eEFSec to deliver Sec. eEFSec and SBP2 do not interact directly but rather deploy their carboxyl-terminal domains to engage with the opposite ends of the SECIS. By using its Lys-rich and carboxyl-terminal segments, the ribosomal protein eS31 simultaneously interacts with Sec-specific transfer RNA (tRNASec) and SBP2, which further stabilizes the assembly. eEFSec is indiscriminate toward l-serine and facilitates its misincorporation at Sec UGA codons. Our results support a fundamentally distinct mechanism of Sec UGA recoding in eukaryotes from that in bacteria

Molecular weight of hydroxyethyl starch: is there an effect on blood coagulation and pharmacokinetics?

Background. The development of hydroxyethyl starches (HES) with low impact on blood coagulation but higher volume effect compared with the currently used HES solutions is of clinical interest. We hypothesized that high molecular weight, low-substituted HES might possess these properties. Methods. Thirty pigs were infused with three different HES solutions (20 ml kg−1) with the same degree of molar substitution (0.42) but different molecular weights (130, 500 and 900 kDa). Serial blood samples were taken over 24 h and blood coagulation was assessed by Thromboelastograph® analysis and analysis of plasma coagulation. In addition, plasma concentration and in vivo molecular weight were determined and pharmacokinetic data were computed based on a two-compartment model. Results. Thromboelastograph analysis and plasma coagulation tests did not reveal a more pronounced alteration of blood coagulation with HES 500 and HES 900 compared with HES 130. In contrast, HES 500 and HES 900 had a greater area under the plasma concentration-time curve [1542 (142) g min litre−1, P<0.001, 1701 (321) g min litre−1, P<0.001] than HES 130 [1156 (223) g min litre−1] and alpha half life (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} $\mathrm{t}_{{\alpha}}^{{\frac{1}{2}}}$ \end{document}) was longer for HES 500 [53.8 (8.6) min, P<0.01] and HES 900 [57.1 (12.3) min, P<0.01] than for HES 130 [39.9 (10.7) min]. Beta half life (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} $\mathrm{t}_{{\beta}}^{{\frac{1}{2}}}$ \end{document}), however, was similar for all three types of HES [from 332 (100) to 381 (63) min]. Conclusions. In low-substituted HES, molecular weight is not a key factor in compromising blood coagulation. The longer initial intravascular persistence of high molecular weight low-substituted HES might result in a longer lasting volume effec

Divergent architecture of the heterotrimeric NatC complex explains N-terminal acetylation of cognate substrates

The heterotrimeric NatC complex, comprising the catalytic Naa30 and the two auxiliary subunits Naa35 and Naa38, co-translationally acetylates the N-termini of numerous eukaryotic target proteins. Despite its unique subunit composition, its essential role for many aspects of cellular function and its suggested involvement in disease, structure and mechanism of NatC have remained unknown. Here, we present the crystal structure of the Saccharomyces cerevisiae NatC complex, which exhibits a strikingly different architecture compared to previously described N-terminal acetyltransferase (NAT) complexes. Cofactor and ligand-bound structures reveal how the first four amino acids of cognate substrates are recognized at the Naa30–Naa35 interface. A sequence-specific, ligand-induced conformational change in Naa30 enables efficient acetylation. Based on detailed structure–function studies, we suggest a catalytic mechanism and identify a ribosome-binding patch in an elongated tip region of NatC. Our study reveals how NAT machineries have divergently evolved to N-terminally acetylate specific subsets of target proteins

Nat Struct Mol Biol

Internal ribosome entry sites (IRESs) facilitate an alternative, end-independent pathway of translation initiation. A particular family of dicistroviral IRESs can assemble elongation-competent 80S ribosomal complexes in the absence of canonical initiation factors and initiator transfer RNA. We present here a cryo-EM reconstruction of a dicistroviral IRES bound to the 80S ribosome. The resolution of the cryo-EM reconstruction, in the subnanometer range, allowed the molecular structure of the complete IRES in its active, ribosome-bound state to be solved. The structure, harboring three pseudoknot-containing domains, each with a specific functional role, shows how defined elements of the IRES emerge from a compactly folded core and interact with the key ribosomal components that form the A, P and E sites, where tRNAs normally bind. Our results exemplify the molecular strategy for recruitment of an IRES and reveal the dynamic features necessary for internal initiation

Preconditioning with sevoflurane decreases PECAM-1 expression and improves one-year cardiovascular outcome in coronary artery bypass graft surgery

Background. Cardiac preconditioning is thought to be involved in the observed decreased coronary artery reocclusion rate in patients with angina preceding myocardial infarction. We prospectively examined whether preconditioning by sevoflurane would decrease late cardiac events in patients undergoing coronary artery bypass graft (CABG) surgery. Methods. Seventy-two patients scheduled for elective CABG surgery were randomized to preconditioning by sevoflurane (10 min at 4 vol%) or placebo. For all patients, follow-up of adverse cardiac events was obtained 6 and 12 months after surgery. Transcript levels for platelet-endothelial cell adhesion molecule-1 (PECAM-1/CD31), catalase and heat shock protein 70 (Hsp70) were determined in atrial biopsies after sevoflurane preconditioning. Results. Pharmacological preconditioning by sevoflurane reduced the incidence of late cardiac events during the first year after CABG surgery (sevoflurane 3% vs 17% in the placebo group, log-rank test, P=0.038). One patient in the sevoflurane group and three patients in the placebo group experienced new episodes of congestive heart failure and three additional patients had coronary artery reocclusion. Perioperative peak concentrations for myocardial injury markers were higher in patients with subsequent late cardiac events [NTproBNP, 9031 (4125) vs 3049 (1906) ng litre−1, P<0.001; cTnT, 1.31 (0.88) vs 0.46 (0.29) µg litre−1, P<0.001]. Transcript levels were reduced for PECAM-1 and increased for catalase but unchanged for Hsp70 in atrial biopsies after sevoflurane preconditioning. Conclusions. This prospective randomized clinical study provides evidence of a protective role for pharmacological preconditioning by sevoflurane in late cardiac events in CABG patients, which may be related to favourable transcriptional changes in pro- and antiprotective protein

Ire1α-Regulated Rate of mRNA Translation is Required for Acquisition of Identity and Polarity in Upper Layer Cortical Neurons

Evolutionary expansion of the neocortex is associated with the increase in upper layer neurons. Here, we present Inositol-Requiring Enzyme 1α, Ire1α, as an essential determinant of upper layer fate, neuronal polarization and cortical lamination. We demonstrate a non-canonical function of Ire1α in the regulation of global translation rates in the developing neocortex through its dynamic interaction with the ribosome and regulation of eIF4A1 and eEF-2 expression. Inactivation of Ire1α engenders lower protein synthesis rates associated with stalled ribosomes and decreased number of translation start sites. We show unique sensitivity of upper layer fate to translation rates. Whereas eEF-2 is required for cortical lamination, eIF4A1 regulates acquisition of upper layer fate downstream of Ire1α in a mechanism of translational control dependent on 5’UTR-embedded structural elements in fate determinant genes. Our data unveil developmental regulation of ribosome dynamics as post-transcriptional mechanisms orchestrating neuronal diversity establishment and assembly of cortical layers

Molar substitution and C2/C6 ratio of hydroxyethyl starch: influence on blood coagulation

Background. Development of hydroxyethyl starches (HES) with a low impact on blood coagulation but a long intravascular persistence is of clinical interest. A previous in vitro study showed that low substituted high molecular weight HES does not compromise blood coagulation more than medium molecular weight HES. In the present study we assessed the individual effects on blood coagulation of molar substitution and C2/C6 ratio of a high molecular weight HES. Methods. Blood was obtained from 30 healthy patients undergoing elective surgery and mixed with six high molecular weight (700 kDa) HES solutions differing in their molar substitution (0.42 and 0.51) and C2/C6 ratio (2.7, 7 and 14) to achieve 20, 40 and 60% dilution. Blood coagulation was assessed by Thrombelastograph® analysis (TEG) and plasma coagulation tests.Data were compared using a three-way analysis of variance model with repeated measures on the three factors. Results. Higher molar substitution compromised blood coagulation most (for all TEG parameters, P0.50). The higher molar substitution was associated with a lesser increase in PT (P=0.007) and a greater decrease in factor VIII (P=0.010). PTT, functional and antigenic von Willebrand factors were not significantly influenced by molar substitution (P for all >0.20). No significant differences between solutions with the same molar substitution but different C2/C6 ratios were found in plasma coagulation parameters (P for all >0.05). Conclusions. TEG analysis indicates that high molecular HES with a molar substitution of 0.42 and a C2/C6 ratio of 2.7 has the lowest effect on in vitro human blood coagulatio