Saccharomyces cerevisiae Ngl3p is an active 3'-5' exonuclease with a specificity towards poly-A RNA reminiscent of cellular deadenylases

Deadenylation is the first and rate-limiting step during turnover of mRNAs in eukaryotes. In the yeast, Saccharomyces cerevisiae, two distinct 3'-5' exonucleases, Pop2p and Ccr4p, have been identified within the Ccr4-NOT deadenylase complex, belonging to the DEDD and Exonuclease-Endonuclease-Phosphatase (EEP) families, respectively. Ngl3p has been identified as a new member of the EEP family of exonucleases based on sequence homology, but its activity and biological roles are presently unknown. Here, we show using in vitro deadenylation assays on defined RNA species mimicking poly-A containing mRNAs that yeast Ngl3p is a functional 3'-5' exonuclease most active at slightly acidic conditions. We further show that the enzyme depends on divalent metal ions for activity and possesses specificity towards poly-A RNA similar to what has been observed for cellular deadenylases. The results suggest that Ngl3p is naturally involved in processing of poly-adenylated RNA and provide insights into the mechanistic variations observed among the redundant set of EEP enzymes found in yeast and higher eukaryotes.     

Zur Frage des Einflusses von Spurenelementen auf Bacillus asterosporus

Zusammenfassung Es wurde mittels Trübungsmessung der Einfluß von Chloriden, Nitraten und Sulfaten der Schwermetalle Kupfer, Zink und Kobalt auf das Wachstum von Bacillus asterosporus im Konzentrationsbereich von 1–14·10^-7 mol/ml synthetischer Nährlösung geprüft. Dabei konnte nur durch Kobalt in hoher Verdünnung eine Wachstumsförderung bis zu 100% erzielt werden, während die beiden anderen Schwermetalle toxisch wirkten. Der Einfluß der gleichzeitig vorhandenen Anionen erscheint dabei von untergeordneter Bedeutung. Mit Bor, als Borax dem Nährmedium zugesetzt, konnte im Konzentrationsbereich von 2·10^-7 Mol/ml auch eine Wachstumsförderung erzielt werden.Auf chemischem und radiochemischem Wege wurde schließlich übereinstimmend festgestellt, daß Bac. asterosporus im Bereich der das Wachstum am stärksten anregenden Konzentration von Co(NO₃)₂ eine bestimmte Kobaltmenge adsorptiv oder intracellulär festhält. Die Frage nach dem Sitz des fixierten Kobalt konnte nicht eindeutig geklärt werden.Über Versuche zur Klärung des Kobalteinflusses auf den Abbau von Glucose und die Abgabe von Gärungskohlensäure durch Bacillus asterosporus wird demnächst berichtet werden.     

Role of sequence and position of the cleavage sites in prothrombin activation

In the penultimate step of the coagulation cascade, the multidomain vitamin-K-dependent zymogen prothrombin is converted to thrombin by the prothrombinase complex composed of factor Xa, cofactor Va, and phospholipids. Activation of prothrombin requires cleavage at two residues, R271 and R320, along two possible pathways generating either the intermediate prethrombin-2 (following initial cleavage at R271) or meizothrombin (following initial cleavage at R320). The former pathway is preferred in the absence of and the latter in the presence of cofactor Va. Several mechanisms have been proposed to explain this preference, but the role of the sequence and position of the sites of cleavage has not been thoroughly investigated. In this study, we engineered constructs where the sequences ²⁶¹DEDSDRAIEGRTATSEYQT²⁷⁹ and ³¹⁰RELLESYIDGRIVEGSDAE³²⁸ were swapped between the R271 and R320 sites. We found that in the absence of cofactor Va, the wild-type sequence at the R271 site is cleaved preferentially regardless of its position at the R271 or R320 site, whereas in the presence of cofactor Va, the R320 site is cleaved preferentially regardless of its sequence. Additional single-molecule FRET measurements revealed that the environment of R271 changes significantly upon cleavage at R320 due to the conformational transition from the closed form of prothrombin to the open form of meizothrombin. Detailed kinetics of cleavage at the R271 site were monitored by a newly developed assay based on loss of FRET. These findings show how sequence and position of the cleavage sites at R271 and R320 dictate the preferred pathway of prothrombin activation.     

Introduction of Pseudomonas aeruginosa into a Hospital via Vegetables

Pseudomonas aeruginosa was isolated from tomatoes, radishes, celery, carrots, endive, cabbage, cucumbers, onions, and lettuce obtained from the kitchen of a general hospital, with tomatoes yielding both highest frequencies of isolation and highest counts. Presence of P. aeruginosa on the hands of kitchen personnel and cutting boards and knives which they used suggests acquisition of the organism through contact with these vegetables. It is estimated that a patient consuming an average portion of tomato salad might ingest as many as 5 x 10(3) colony-forming units of P. aeruginosa. Pyocine types of P. aeruginosa isolated from clinical specimens were frequently identical to those recovered from vegetables, thus implicating tomatoes and other vegetables as an important source and vehicle by which P. aeruginosa colonizes the intestinal tract of patients.     

Coronary CT angiography for suspected acute coronary syndrome: sex-associated differences

AimThe optimal diagnostic test in the work-up of suspected acute coronary syndrome (ACS) may differ between men and women. The aim of this study was to compare sex-associated differences between using a diagnostic strategy including early coronary computed tomography angiography (CCTA) and standard of care (SOC).MethodsIn total, 500 patients who presented with symptoms suggestive of ACS at the emergency department were randomised between a diagnostic strategy supplemented with early CCTA and SOC.ResultsWomen were generally older than men (mean ± standard deviation 56 ± 10 vs 53 ± 10 years, p < 0.01) and were less often admitted to hospital (33% vs 44%, p = 0.02). Obstructive coronary artery disease on CCTA (> 50% luminal narrowing) was less frequently seen in women (14% vs 26%, p = 0.02), and ACS was diagnosed less often in women (5% vs 10%, p = 0.03). Women underwent less outpatient testing when early CCTA was used in the emergency department evaluation of suspected ACS (p = 0.008).ConclusionWomen had a lower incidence of obstructive CAD on CCTA and were less often admitted to hospital than men. They were subjected to less outpatient testing when early CCTA was used in the emergency department evaluation of suspected ACS.Supplementary InformationThe online version of this article (10.1007/s12471-021-01607-1) contains supplementary material, which is available to authorized users.