Biochemical characterisation of class III biotin protein ligases from Botrytis cinerea and Zymoseptoria tritici.

Biotin protein ligase (BPL) is an essential enzyme in all kingdoms of life, making it a potential target for novel anti-infective agents. Whilst bacteria and archaea have simple BPL structures (class I and II), the homologues from certain eukaryotes such as mammals, insects and yeast (class III) have evolved a more complex structure with a large extension on the N-terminus of the protein in addition to the conserved catalytic domain. The absence of atomic resolution structures of any class III BPL hinders structural and functional analysis of these enzymes. Here, two new class III BPLs from agriculturally important moulds Botrytis cinerea and Zymoseptoria tritici were characterised alongside the homologue from the prototypical yeast Saccharomyces cerevisiae. Circular dichroism and ion mobility-mass spectrometry analysis revealed conservation of the overall tertiary and secondary structures of all three BPLs, corresponding with the high sequence similarity. Subtle structural differences were implied by the different thermal stabilities of the enzymes and their varied Michaelis constants for their interactions with ligands biotin, MgATP, and biotin-accepting substrates from different species. The three BPLs displayed different preferences for fungal versus bacterial protein substrates, providing further evidence that class III BPLs have a 'substrate validation' activity for selecting only appropriate proteins for biotinylation. Selective, potent inhibition of these three BPLs was demonstrated despite sequence and structural homology. This highlights the potential for targeting BPL for novel, selective antifungal therapies against B. cinerea, Z. tritici and other fungal species.

Optimization of drug therapy in elderly individuals admitted to a geriatric unit.

BACKGROUND: A substantial share of adverse drug events involves inappropriate prescribing (IP). Specialized geriatric units are supposed to pay particular attention to prescribing appropriateness and to promoting a higher prescribing quality. OBJECTIVE: The objective of this study was to evaluate the reality of such assessment and optimization in real life (usual care) in a population of elderly individuals admitted to a geriatric unit. METHOD: This is an observational study including all older patients admitted to an acute geriatric unit over a 6-month period. As part of usual care, the geriatrician is supposed to detect potentially inappropriate medication and potential prescribing omission using validated tools. The primary outcome was the prevalence rate of therapeutic modifications motivated by treatment optimization (stop, switch, or introduction). Multivariate logistic regression analyses were performed to identify the factors associated with therapeutic discontinuation. RESULTS: A total of 216 patients were included. The mean age was 85.7 years. Included patients had an average of 7.2±3.3 drugs at admission and 5.8±2.7 at discharge. IP was highly prevalent in our study where about 63% of the patients had experienced at least one modification because of overuse. The most commonly discontinued medications were drugs used to treat gastroesophageal reflux disease and peptic ulcer disease and serotonin reuptake inhibitor antidepressants. The most commonly introduced medications were analgesics and warfarin. By using multivariate analysis, we found that patient age and number of drugs on admission were significantly associated with medication discontinuation during hospital stay. CONCLUSION: In this real-life study of all patients admitted to a Geriatric Post Emergency Unit, 83% of the patients had a treatment modification during hospital stay. The most original result of our study is the clear reduction in polypharmacy during hospitalization.

Production and secretion of a heterologous protein by turnip hairy roots with superiority over tobacco hairy roots.

A fully contained and efficient heterologous protein production system was designed using Brassica rapa rapa (turnip) hairy roots. Two expression cassettes containing a cauliflower mosaic virus (CaMV) 35S promoter with a duplicated enhancer region, an Arabidopsis thaliana sequence encoding a signal peptide and the CaMV polyadenylation signal were constructed. One cassette was used to express the green fluorescent protein (GFP)-encoding gene in hairy roots grown in flasks. A stable and fast-growing hairy root line secreted GFP at >120 mg/l culture medium. GFP represented 60 % of the total soluble proteins in the culture medium. Turnip hairy roots retained sustainable growth and stable GFP production over 3 years. These results were superior to those obtained using tobacco hairy roots.

Cloning, expression and characterization of insulin-degrading enzyme from tomato (Solanum lycopersicum).

A cDNA encoding insulin-degrading enzyme (IDE) was cloned from tomato (Solanum lycopersicum) and expressed in Escherichia coli in N-terminal fusion with glutathione S-transferase. GST-SlIDE was characterized as a neutral thiol-dependent metallopeptidase with insulinase activity: the recombinant enzyme cleaved the oxidized insulin B chain at eight peptide bonds, six of which are also targets of human IDE. Despite a certain preference for proline in the vicinity of the cleavage site, synthetic peptides were cleaved at apparently stochastic positions indicating that SlIDE, similar to IDEs from other organisms, does not recognize any particular amino acid motif in the primary structure of its substrates. Under steady-state conditions, an apparent K(m) of 62+/-7 microm and a catalytic efficiency (k(cat)/K(m)) of 62+/-15 mm(-1) s(-1) were determined for Abz-SKRDPPKMQTDLY(NO(3))-NH(2) as the substrate. GST-SlIDE was effectively inhibited by ATP at physiological concentrations, suggesting regulation of its activity in response to the energy status of the cell. While mammalian and plant IDEs share many of their biochemical properties, this similarity does not extend to their function in vivo, because insulin and the beta-amyloid peptide, well-established substrates of mammalian IDEs, as well as insulin-related signaling appear to be absent from plant systems.