The Pro-Apoptotic BH3-Only Protein Bim Interacts with Components of the Translocase of the Outer Mitochondrial Membrane (TOM)

The pro-apoptotic Bcl-2-family protein Bim belongs to the BH3-only proteins known as initiators of apoptosis. Recent data show that Bim is constitutively inserted in the outer mitochondrial membrane via a C-terminal transmembrane anchor from where it can activate the effector of cytochrome c-release, Bax. To identify regulators of Bim-activity, we conducted a search for proteins interacting with Bim at mitochondria. We found an interaction of Bim with Tom70, Tom20 and more weakly with Tom40, all components of the Translocase of the Outer Membrane (TOM). In vitro import assays performed on tryptically digested yeast mitochondria showed reduced Bim insertion into the outer mitochondrial membrane (OMM) indicating that protein receptors may be involved in the import process. However, RNAi against components of TOM (Tom40, Tom70, Tom22 or Tom20) by siRNA, individually or in combination, did not consistently change the amount of Bim on HeLa mitochondria, either at steady state or upon de novo-induction. In support of this, the individual or combined knock-downs of TOM receptors also failed to alter the susceptibility of HeLa cells to Bim-induced apoptosis. In isolated yeast mitochondria, lack of Tom70 or the TOM-components Tom20 or Tom22 alone did not affect the import of Bim into the outer mitochondrial membrane. In yeast, expression of Bim can sensitize the cells to Bax-dependent killing. This sensitization was unaffected by the absence of Tom70 or by an experimental reduction in Tom40. Although thus the physiological role of the Bim-TOM-interaction remains unclear, TOM complex components do not seem to be essential for Bim insertion into the OMM. Nevertheless, this association should be noted and considered when the regulation of Bim in other cells and situations is investigated.     

The female postabdomen of the enigmatic Nannochoristidae (Insecta: Mecopterida) and its phylogenetic significance

Hünefeld, F. and Beutel, R.G. 2011. The female postabdomen of the enigmatic Nannochoristidae (Insecta: Mecopterida) and its phylogenetic significance. —Acta Zoologica (Stockholm) 00: 1–8. External and internal features of the female postabdomen of Nannochorista neotropica are described in detail. The conditions found in females of Nannochoristidae come closest to the ground plan of Mecopterida. This lineage is characterised by telescoping postabdominal segments, a presumptive autapomorphic feature that is modified in some antliophoran groups, but displayed by the nannochoristid species in a typical manner. More potential autapomorphies of Mecopterida, all present in Nannochoristidae, are the neo‐formation of an intersegmental muscle, a transverse muscle spanning between the genital appendages of segment VIII, a muscle connecting these appendages and the genital chamber and the loss of an intersegmental muscle. Plesiomorphic features of Nannochoristidae are the presence of paired genital appendages on segments VIII and IX. Information on the egg‐depositing substrates of the females is not available. The telescoping postabdomen is suitable for oviposition in soft substrates such as moist soil, or rotten plant materials in the riparian zone, and this is possibly a ground‐plan feature of Mecopterida. The results of recent phylogenetic analyses based on morphological data support a placement of Nannochoristidae in Antliophora, whereas the exact position of the group remains ambiguous. No characters of the female postabdomen were found supporting the monophyly of Mecoptera as conventionally circumscribed, that is Nannochoristidae + Boreidae + Pistillifera.     

Heterocyclic methylsulfone hydroxamic acid LpxC inhibitors as Gram-negative antibacterial agents

The synthesis and antibacterial activity of heterocyclic methylsulfone hydroxamates is presented. Compounds in this series are potent inhibitors of the LpxC enzyme, a key enzyme involved in the production of lipopolysaccharide (LPS) found in the outer membrane of Gram-negative bacteria. SAR evaluation of compounds in this series revealed analogs with potent antibacterial activity against challenging Gram-negative species such as Pseudomonas aeruginosa and Klebsiella pneumoniae.     

Aryl Hetaryl Ketones and Thioketones as Efficient Inhibitors of Peptidyl‐Prolyl cis‐trans Isomerases

A series of 18 differently substituted new aryl hetaryl ketones and thioketones were synthesized in four to six steps from commercial starting materials. The new ketones were evaluated as inhibitors of the peptidyl‐prolyl cis‐trans isomerase hPin1 with K_i values ranging in the one‐digit micromolar to sub‐micromolar numbers. A crystal structure revealed the non‐planar arrangement of the aryl residues at the carbonyl compound and supports the hypothesis that the new compounds might mimic the transition state of the enzymatic conversion.     

Surgical stabilisation of the spine compared with a programme of intensive rehabilitation for the management of patients with chronic low back pain: cost utility analysis based on a randomised controlled trial

Objective To determine whether, from a health provider and patient perspective, surgical stabilisation of the spine is cost effective when compared with an intensive programme of rehabilitation in patients with chronic low back pain.Design Economic evaluation alongside a pragmatic randomised controlled trial.Setting Secondary care.Participants 349 patients randomised to surgery (n = 176) or to an intensive rehabilitation programme (n = 173) from 15 centres across the United Kingdom between June 1996 and February 2002.Main outcome measures Costs related to back pain and incurred by the NHS and patients up to 24 months after randomisation. Return to paid employment and total hours worked. Patient utility as estimated by using the EuroQol EQ-5D questionnaire at several time points and used to calculate quality adjusted life years (QALYs). Cost effectiveness was expressed as an incremental cost per QALY.Results At two years, 38 patients randomised to rehabilitation had received rehabilitation and surgery whereas just seven surgery patients had received both treatments. The mean total cost per patient was estimated to be £7830 (SD £5202) in the surgery group and £4526 (SD £4155) in the intensive rehabilitation arm, a significant difference of £3304 (95% confidence interval £2317 to £4291). Mean QALYs over the trial period were 1.004 (SD 0.405) in the surgery group and 0.936 (SD 0.431) in the intensive rehabilitation group, giving a non-significant difference of 0.068 (–0.020 to 0.156). The incremental cost effectiveness ratio was estimated to be £48 588 per QALY gained (–£279 883 to £372 406).Conclusion Two year follow-up data show that surgical stabilisation of the spine may not be a cost effective use of scarce healthcare resources. However, sensitivity analyses show that this could change—for example, if the proportion of rehabilitation patients requiring subsequent surgery continues to increase.     

Transformation of Claviceps purpurea using a bleomycin resistance gene

Summary To develop a DNA-mediated transformation system for Claviceps purpurea a vector was constructed using a bleomycin-resistance gene (bleo ^R) fused in frame to the Aspergillus nidulans trp C promoter as a dominant selection marker. The construct was shown to be functional in Aspergillus nidulans and Aspergillus niger and used to transform a wild strain of Claviceps purpurea. Transformats were obtained at low frequencies; they were shown to contain transforming DNA integrated into the chromosomal DNA, probably in multimeric copies and at multiple sites. Combined Southern, Northern and resistance level analysis indicate that the A. nidulans promoter is functional in C. purpurea.Dedicated to Professor Dr. Dr. h. c. K. Esser on the occasion of his 65^th birthday     

Mechanistic link between PKR dimerization, autophosphorylation, and eIF2alpha substrate recognition

The antiviral protein kinase PKR inhibits protein synthesis by phosphorylating the translation initiation factor eIF2alpha on Ser51. Binding of double-stranded RNA to the regulatory domains of PKR promotes dimerization, autophosphorylation, and the functional activation of the kinase. Herein, we identify mutations that activate PKR in the absence of its regulatory domains and map the mutations to a recently identified dimerization surface on the kinase catalytic domain. Mutations of other residues on this surface block PKR autophosphorylation and eIF2alpha phosphorylation, while mutating Thr446, an autophosphorylation site within the catalytic-domain activation segment, impairs eIF2alpha phosphorylation and viral pseudosubstrate binding. Mutational analysis of catalytic-domain residues preferentially conserved in the eIF2alpha kinase family identifies helix alphaG as critical for the specific recognition of eIF2alpha. We propose an ordered mechanism of PKR activation in which catalytic-domain dimerization triggers Thr446 autophosphorylation and specific eIF2alpha substrate recognition.     

Roscovitine targets, protein kinases and pyridoxal kinase

(R)-Roscovitine (CYC202) is often referred to as a "selective inhibitor of cyclin-dependent kinases." Besides its use as a biological tool in cell cycle, neuronal functions, and apoptosis studies, it is currently evaluated as a potential drug to treat cancers, neurodegenerative diseases, viral infections, and glomerulonephritis. We have investigated the selectivity of (R)-roscovitine using three different methods: 1) testing on a wide panel of purified kinases that, along with previously published data, now reaches 151 kinases; 2) identifying roscovitine-binding proteins from various tissue and cell types following their affinity chromatography purification on immobilized roscovitine; 3) investigating the effects of roscovitine on cells deprived of one of its targets, CDK2. Altogether, the results show that (R)-roscovitine is rather selective for CDKs, in fact most kinases are not affected. However, it binds an unexpected, non-protein kinase target, pyridoxal kinase, the enzyme responsible for phosphorylation and activation of vitamin B6. These results could help in interpreting the cellular actions of (R)-roscovitine but also in guiding the synthesis of more selective roscovitine analogs.