Centrosomal localization of cyclins E and A: Structural similarities and functional differences

Recent identification of the modular CLS motifs responsible for cyclins A and E localization on centrosomes has revealed a tight linkage between the nuclear and centrosomal cycles. These G₁/S cyclins must localize on the centrosome in order for DNA replication to occur in the nucleus, whereas essential DNA replication factors also function on the centrosome to prevent centrosome overduplication. Both events are dependent on the presence of an intact CLS within each cyclin. Here we compare the cyclins A and E CLSs at the structural and functional levels and identify a new cyclin A CLS mutant that disrupts all CLS functions and reduces the affinity of cyclin A for Cdk2. Analysis of interactions of the CLS motif within the cyclin molecules highlights the importance of the cyclin CBOX1 region for Cdk2 binding.Key words: cyclin A, cyclin E, Cdk2, centrosome, CLS, PSTAIRE, DNA synthesis     

A Canadian Critical Care Trials Group project in collaboration with the international forum for acute care trialists - Collaborative H1N1 Adjuvant Treatment pilot trial (CHAT): study protocol and design of a randomized controlled trial

Background

The Vaccine Assessment using Linked Data (VALiD) trial compared opt-in and opt-out parental consent for a population-based childhood vaccine safety surveillance program using data linkage. A subsequent telephone interview of all households enrolled in the trial elicited parental intent regarding the return or non-return of reply forms for opt-in and opt-out consent. This paper describes the rationale for the trial and provides an overview of the design and methods.

Methods/Design

Single-centre, single-blind, randomised controlled trial (RCT) stratified by firstborn status. Mothers who gave birth at one tertiary South Australian hospital were randomised at six weeks post-partum to receive an opt-in or opt-out reply form, along with information explaining data linkage. The primary outcome at 10 weeks post-partum was parental participation in each arm, as indicated by the respective return or non-return of a reply form (or via telephone or email response). A subsequent telephone interview at 10 weeks post-partum elicited parental intent regarding the return or non-return of the reply form, and attitudes and knowledge about data linkage, vaccine safety, consent preferences and vaccination practices. Enrolment began in July 2009 and 1,129 households were recruited in a three-month period. Analysis has not yet been undertaken. The participation rate and selection bias for each method of consent will be compared when the data are analysed.

Discussion

The VALiD RCT represents the first trial of opt-in versus opt-out consent for a data linkage study that assesses consent preferences and intent compared with actual opting in or opting out behaviour, and socioeconomic factors. The limitations to generalisability are discussed.

Trial registration

Australian New Zealand Clinical Trials Registry ACTRN12610000332022     

Overexpression of human peroxiredoxin 5 in subcellular compartments of Chinese hamster ovary cells: effects on cytotoxicity and DNA damage caused by peroxides

Peroxiredoxin 5 is a mammalian thioredoxin peroxidase ubiquitously expressed in tissues. Peroxiredoxin 5 can be intracellularly localized to mitochondria, peroxisomes, the cytosol, and, to a lesser extent, the nucleus. This remarkably wide subcellular distribution compared with the five other mammalian peroxiredoxins prompted us to further investigate the antioxidant protective function of peroxiredoxin 5 in mammalian cells according to its subcellular localization. Chinese hamster ovary cells overexpressing human peroxiredoxin 5 in the cytosol, in mitochondria, or in the nucleus were established by stable transfection. Cells overexpressing peroxiredoxin 5 were exposed for 1 h to low or acute oxidative stress with exogenously added hydrogen peroxide or tert-butylhydroperoxide. Cell protection conferred by peroxiredoxin 5 was evaluated by clonogenicity and lactate dehydrogenase assays. Overexpressing peroxiredoxin 5 in either the cytosolic, mitochondrial, or nuclear compartment significantly reduced cell death, with more effective protection with overexpression of peroxiredoxin 5 in mitochondria, confirming that this organelle is a major target of peroxides. Moreover, we evaluated, with the comet assay, nuclear DNA damage induced by hydrogen peroxide or tert-butylhydroperoxide. Overexpression of peroxiredoxin 5 in the nucleus significantly decreased DNA damage induced by both peroxides. In conclusion, the present study suggests that multiple subcellular targeting of peroxiredoxin 5 in mammalian cells can be implicated in antioxidant protective mechanisms under nonpathological conditions but also during acute oxidative stress caused by peroxides occurring in pathophysiological situations.     

Structure and function analysis of peptide antagonists of melanoma inhibitor of apoptosis (ML-IAP)

Melanoma inhibitor of apoptosis (ML-IAP) is a potent anti-apoptotic protein that is upregulated in a number of melanoma cell lines but not expressed in most normal adult tissues. Overexpression of IAP proteins, such as ML-IAP or the ubiquitously expressed X-chromosome-linked IAP (XIAP), in human cancers has been shown to suppress apoptosis induced by a variety of stimuli. Peptides based on the processed N-terminus of Smac/DIABLO can negate the ability of overexpressed ML-IAP or XIAP to suppress drug-induced apoptosis. Such peptides have been demonstrated to bind to the single baculovirus IAP repeat (BIR) of ML-IAP and the third BIR of XIAP with similar high affinities (approximately 0.5 microM). Herein, we use phage-display of na?ve peptide libraries and synthetic peptides to investigate the peptide-binding properties of ML-IAP-BIR and XIAP-BIR3. X-ray crystal structures of ML-IAP-BIR in complex with Smac- and phage-derived peptides, together with peptide structure-activity-relationship data, indicate that the peptides can be modified to provide increased binding affinity and selectivity for ML-IAP-BIR relative to XIAP-BIR3. For instance, substitution of Pro3' in the Smac-based peptide (AVPIAQKSE) with (2S,3S)-3-methylpyrrolidine-2-carboxylic acid [(3S)-methyl-proline] results in a peptide with 7-fold greater affinity for ML-IAP-BIR and about 100-fold specificity for ML-IAP-BIR relative to XIAP-BIR3.     

Importing statistical measures into Artemis enhances gene identification in the <Emphasis Type="Italic">Leishmania</Emphasis> genome project

Background

Seattle Biomedical Research Institute (SBRI) as part of the Leishmania Genome Network (LGN) is sequencing chromosomes of the trypanosomatid protozoan species Leishmania major. At SBRI, chromosomal sequence is annotated using a combination of trained and untrained non-consensus gene-prediction algorithms with ARTEMIS, an annotation platform with rich and user-friendly interfaces.

Results

Here we describe a methodology used to import results from three different protein-coding gene-prediction algorithms (GLIMMER, TESTCODE and GENESCAN) into the ARTEMIS sequence viewer and annotation tool. Comparison of these methods, along with the CODON USAGE algorithm built into ARTEMIS, shows the importance of combining methods to more accurately annotate the L. major genomic sequence.

Conclusion

An improvised and powerful tool for gene prediction has been developed by importing data from widely-used algorithms into an existing annotation platform. This approach is especially fruitful in the Leishmania genome project where there is large proportion of novel genes requiring manual annotation.

     

Effects of different dietary regimes alone or in combination with standardized Aronia melanocarpa extract supplementation on lipid and fatty acids profiles in rats

This study investigated different dietary strategies, high-fat (HFd), or standard diet (Sd) alone or in combination with standardized Aronia melanocarpa extract (SAE), as a polyphenol-rich diet, and their effects on lipids and fatty acids (FA) in rats with metabolic syndrome (MetS). Wistar albino rats were randomly divided into two groups: healthy and rats with MetS, and then depending on dietary patterns on six groups: healthy rats fed with Sd, healthy rats fed with Sd and SAE, rats with MetS fed with HFd, rats with MetS fed with HFd and SAE, rats with MetS fed with Sd, and rats with MetS fed with Sd and SAE. 4 weeks later, after an overnight fast (12–14 h), blood for determination of total cholesterol (TC), triglycerides (TG), high-density lipoprotein (HDL), low-density lipoprotein (LDL), index of lipid peroxidation (measured as TBARS), and FA was collected. Increased FA and lipid concentration found in MetS rats were reduced when changing dietary habits from HFd to Sd with or without SAE consumption. Consumption of SAE slightly affects the FA profiles, mostly palmitoleic acid in healthy rats and PUFA in MetS?+?HFd rats. Nevertheless, in a high-fat diet, SAE supplementation significantly decreases n-6/n-3 ratio, thereby decreasing systemic inflammation. Further researches are warranted to confirm these effects in humans.

     

Cortical Dysfunction Underlies the Development of the Split-Hand in Amyotrophic Lateral Sclerosis

The split-hand phenomenon, a specific feature of amyotrophic lateral sclerosis (ALS), refers to preferential wasting of abductor pollicis brevis (APB) and first dorsal interosseous (FDI) with relative preservation of abductor digiti minimi (ADM). The pathophysiological mechanisms underlying the split-hand phenomenon remain elusive and resolution of this issue would provide unique insights into ALS pathophysiology. Consequently, the present study dissected out the relative contribution of cortical and peripheral processes in development of the split-hand phenomenon in ALS. Cortical and axonal excitability studies were undertaken on 26 ALS patients, with motor responses recorded over the APB, FDI and ADM muscles. Results were compared to 21 controls. Short interval intracortical inhibition (SICI), a biomarker of cortical excitability, was significantly reduced across the range of intrinsic hand muscles (APB_{SICI ALS} 0.3±2.0%, APB_{SICI controls} 16.0±1.9%, P<0.0001; FDI_{SICI ALS} 2.7±1.7%, FDI _{SICI controls} 14.8±1.9%, P<0.0001; ADM_{SICI ALS} 2.6±1.5%, ADM _{SICI controls} 9.7±2.2%, P<0.001), although the reduction was most prominent when recorded over APB/FDI. Changes in SICI were accompanied by a significant increase in motor evoked potential amplitude and reduction of cortical silent period duration, all indicative of cortical hyperexcitability, and these were most prominent from the APB/FDI. At a peripheral level, a significant increase in strength-duration time constant and reduction in depolarising threshold electrotonus were evident in ALS, although these changes did not follow a split-hand distribution. Cortical dysfunction contributed to development of the split-hand in ALS, thereby implying an importance of cortical hyperexcitability in ALS pathogenesis.     

cIAP1 and TAK1 protect cells from TNF-induced necrosis by preventing RIP1/RIP3-dependent reactive oxygen species production

Three members of the IAP family (X-linked inhibitor of apoptosis (XIAP), cellular inhibitor of apoptosis proteins-1/-2 (cIAP1 and cIAP2)) are potent suppressors of apoptosis. Recent studies have shown that cIAP1 and cIAP2, unlike XIAP, are not direct caspase inhibitors, but block apoptosis by functioning as E3 ligases for effector caspases and receptor-interacting protein 1 (RIP1). cIAP-mediated polyubiquitination of RIP1 allows it to bind to the pro-survival kinase transforming growth factor-β-activated kinase 1 (TAK1) which prevents it from activating caspase-8-dependent death, a process reverted by the de-ubiquitinase CYLD. RIP1 is also a regulator of necrosis, a caspase-independent type of cell death. Here, we show that cells depleted of the IAPs by treatment with the IAP antagonist BV6 are greatly sensitized to tumor necrosis factor (TNF)-induced necrosis, but not to necrotic death induced by anti-Fas, poly(I:C) oxidative stress. Specific targeting of the IAPs by RNAi revealed that repression of cIAP1 is responsible for the sensitization. Similarly, lowering TAK1 levels or inhibiting its kinase activity sensitized cells to TNF-induced necrosis, whereas repressing CYLD had the opposite effect. We show that this sensitization to death is accompanied by enhanced RIP1 kinase activity, increased recruitment of RIP1 to Fas-associated via death domain and RIP3 (which allows necrosome formation), and elevated RIP1 kinase-dependent accumulation of reactive oxygen species (ROS). In conclusion, our data indicate that cIAP1 and TAK1 protect cells from TNF-induced necrosis by preventing RIP1/RIP3-dependent ROS production.