The redox-Bohr group associated with iron-sulfur cluster N2 of complex I

Proton pumping respiratory complex I (NADH: ubiquinone oxidoreductase) is a major component of the oxidative phosphorylation system in mitochondria and many bacteria. In mammalian cells it provides 40% of the proton motive force needed to make ATP. Defects in this giant and most complicated membrane-bound enzyme cause numerous human disorders. Yet the mechanism of complex I is still elusive. A group exhibiting redox-linked protonation that is associated with iron-sulfur cluster N2 of complex I has been proposed to act as a central component of the proton pumping machinery. Here we show that a histidine in the 49-kDa subunit that resides near iron-sulfur cluster N2 confers this redox-Bohr effect. Mutating this residue to methionine in complex I from Yarrowia lipolytica resulted in a marked shift of the redox midpoint potential of iron-sulfur cluster N2 to the negative and abolished the redox-Bohr effect. However, the mutation did not significantly affect the catalytic activity of complex I and protons were pumped with an unchanged stoichiometry of 4 H+/2e(-). This finding has significant implications on the discussion about possible proton pumping mechanism for complex I.

On the derived category of a regular toric scheme

Let X be a quasi-compact scheme, equipped with an open covering by affine schemes U s = Spec A s . A quasi-coherent sheaf on X gives rise, by taking sections over the U s , to a diagram of modules over the coordinate rings A s , indexed by the intersection poset S of the covering. If X is a regular toric scheme over an arbitrary commutative ring, we prove that the unbounded derived category of quasi-coherent sheaves on X can be obtained from a category of Sop-diagrams of chain complexes of modules by inverting maps which induce homology isomorphisms on hyper-derived inverse limits. Moreover, we show that there is a finite set of weak generators, one for each cone in the fan S. The approach taken uses the machinery of Bousfield–Hirschhorn colocalisation of model categories. The first step is to characterise colocal objects; these turn out to be homotopy sheaves in the sense that chain complexes over different open sets U s agree on intersections up to quasi-isomorphism. In a second step it is shown that the homotopy category of homotopy sheaves is equivalent to the derived category of X.

GERMAN WORKS COUNCILS AND THE ANATOMY OF WAGES

Using matched employer-employee data from the German LIAB for 2001, the authors found that German works councils are in general associated with higher earnings, even after accounting for establishment- and worker heterogeneity. Works Council wage premia exceed those of collective bargaining and are higher, in fact, where both institutions are present in the workplace. The authors also found evidence indicating that works councils benefit women relative to men and appear to favor foreign, east-German, and service-sector workers as well. Separate evidence from quantile regressions suggests that the conjunction of works council presence and collective bargaining is important to the narrowing process. In smaller plants even the presence of a works council markup depends on the coexistence of the works council entity With the machinery of collective bargaining.

HTS Motors in Aircraft Propulsion: Design Considerations

Current high temperature superconducting (HTS) wires exhibit high current densities enabling their use in electrical rotating machinery. The possibility of designing high power density superconducting motors operating at reasonable temperatures allows for new applications in mobile systems in which size and weight represent key design parameters. Thus, all-electric aircrafts represent a promising application for HTS motors. The design of such a complex system as an aircraft consists of a multi-variable optimization that requires computer models and advanced design procedures. This paper presents a specific sizing model of superconducting propulsion motors to be used in aircraft design. The model also takes into account the cooling system. The requirements for this application are presented in terms of power and dynamics as well as a load profile corresponding to a typical mission. We discuss the design implications of using a superconducting motor on an aircraft as well as the integration of the electrical propulsion in the aircraft, and the scaling laws derived from physics-based modeling of HTS motors.

Cold-induced silencing by long antisense transcripts of an Arabidopsis Polycomb target.

Transcription in eukaryotic genomes generates an extensive array of non-protein-coding RNA, the functional significance of which is mostly unknown. We are investigating the link between non-coding RNA and chromatin regulation through analysis of FLC - a regulator of flowering time in Arabidopsis and a target of several chromatin pathways. Here we use an unbiased strategy to characterize non-coding transcripts of FLC and show that sense/antisense transcript levels correlate in a range of mutants and treatments, but change independently in cold-treated plants. Prolonged cold epigenetically silences FLC in a Polycomb-mediated process called vernalization. Our data indicate that upregulation of long non-coding antisense transcripts covering the entire FLC locus may be part of the cold-sensing mechanism. Induction of these antisense transcripts occurs earlier than, and is independent of, other vernalization markers and coincides with a reduction in sense transcription. We show that addition of the FLC antisense promoter sequences to a reporter gene is sufficient to confer cold-induced silencing of the reporter. Our data indicate that cold-induced FLC antisense transcripts have an early role in the epigenetic silencing of FLC, acting to silence FLC transcription transiently. Recruitment of the Polycomb machinery then confers the epigenetic memory. Antisense transcription events originating from 3' ends of genes might be a general mechanism to regulate the corresponding sense transcription in a condition/stage-dependent manner.

Altered interactions within FY/AtCPSF complexes required for Arabidopsis FCA-mediated chromatin silencing.

The role of RNA metabolism in chromatin silencing is now widely recognized. We have studied the Arabidopsis RNA-binding protein FCA that down-regulates an endogenous floral repressor gene through a chromatin mechanism involving histone demethylase activity. This mechanism needs FCA to interact with an RNA 3' processing/polyadenylation factor (FY/Pfs2p), but the subsequent events leading to chromatin changes are unknown. Here, we show that this FCA-FY interaction is required for general chromatin silencing roles where hairpin transgenes induce DNA methylation of an endogenous gene. We also show 2 conserved RNA processing factors, AtCPSF100 and AtCPSF160, but not FCA, are stably associated with FY in vivo and form a range of different-sized complexes. A hypomorphic fy allele producing a shorter protein, able to provide some FY functions but unable to interact with FCA, reduces abundance of some of the larger MW complexes. Suppressor mutants, which specifically disrupt the FY motif through which FCA interacts, also lacked these larger complexes. Our data support a model whereby FCA, perhaps after recognition of a specific RNA feature, transiently interacts with FY, an integral component of the canonical RNA 3' processing machinery, changing the interactions of the different RNA processing components. These altered interactions would appear to be a necessary step in this RNA-mediated chromatin silencing.

Protein arginine-methyltransferase-dependent oncogenesis

Enzymes that mediate reversible epigenetic modifications have not only been recognized as key in regulating gene expression(1) and oncogenesis(2,3), but also provide potential targets for molecular therapy(4). Although the methylation of arginine 3 of histone 4 ( H4R3) by protein arginine methyltransferase 1 ( PRMT1) is a critical modification for active chromatin(5,6) and prevention of heterochromatin spread(7), there has been no direct evidence of any role of PRMTs in cancer. Here, we show that PRMT1 is an essential component of a novel Mixed Lineage Leukaemia ( MLL) oncogenic transcriptional complex with both histone acetylation and H4R3 methylation activities, which also correlate with the expression of critical MLL downstream targets. Direct fusion of MLL with PRMT1 or Sam68, a bridging molecule in the complex for PRMT1 interaction, could enhance self-renewal of primary haematopoietic cells. Conversely, specific knockdown of PRMT1 or Sam68 expression suppressed MLL-mediated transformation. This study not only functionally dissects the oncogenic transcriptional machinery associated with an MLL fusion complex, but also uncovers-for the first time-an essential function of PRMTs in oncogenesis and reveals their potential as novel therapeutic targets in human cancer.

Confined site construction: An empirical analysis of factors impacting health and safety management

Purpose – The purpose of this paper is to identify, clarify and tabulate the various managerial issues encountered, to aid in the management of the complex health and safety concerns which occur within a confined construction site environment.

Design/methodology/approach – This is achieved through conducting extensive qualitative and qualitative research in the form of case studies, interviews and questionnaire survey.

Findings – The leading managerial issues in the management of health and safety on a confined construction site are found to be: “Difficulty to move materials around site safely”; “Lack of adequate room for the effective handling of materials”; “Difficulty in ensuring site is tidy and all plant and materials are stored safely”; “Close proximity of individuals to operation of large plant and machinery���; and joint fifth “Difficulty in ensuring proper arrangement and collection of waste materials on-site” along with “Difficulty in controlling hazardous materials and equipment on site”.

Practical implications – The resulting implication for practice of these results can be summarised by identifying that with sustained development of urban centres on a global scale, coupled with the increasing complexity of architectural designs, the majority of on-site project management professionals are faced with the onerous task of completing often intricate designs within a limited spatial environment, under strict health and safety parameters.

Originality/value – The subsequent value of the findings are such that just as on-site management professionals successfully identify the various managerial issues highlighted, the successful management of health and safety on a confined construction site is attainable.

Engineering N-linked protein glycosylation with diverse O antigen lipopolysaccharide structures in Escherichia coli

Campylobacter jejuni has a general N-linked protein glycosylation system that can be functionally transferred to Escherichia coli. In this study, we engineered E. coli cells in a way that two different pathways, protein N-glycosylation and lipopolysaccharide (LPS) biosynthesis, converge at the step in which PglB, the key enzyme of the C. jejuni N-glycosylation system, transfers O polysaccharide from a lipid carrier (undecaprenyl pyrophosphate) to an acceptor protein. PglB was the only protein of the bacterial N-glycosylation machinery both necessary and sufficient for the transfer. The relaxed specificity of the PglB oligosaccharyltransferase toward the glycan structure was exploited to create novel N-glycan structures containing two distinct E. coli or Pseudomonas aeruginosa O antigens. PglB-mediated transfer of polysaccharides might be valuable for in vivo production of O polysaccharides-protein conjugates for use as antibacterial vaccines.

Wzx proteins involved in biosynthesis of O antigen function in association with the first sugar of the O-specific lipopolysaccharide subunit

One of the most common pathways for the export of O-specific lipopolysaccharide (LPS) across the plasma membrane requires the participation of the Wzx protein. Wzx belongs to a family of integral membrane proteins that share little conservation in their primary amino acid sequence, making it difficult to delineate functional domains. This paper reports the cloning and expression in Escherichia coli K-12 of various Wzx homologues from different bacteria as FLAG epitope-tagged protein fusions. A reconstitution system for O16 LPS synthesis was used to assess the ability of each Wzx protein to complement an E. coli K-12 Deltawzx mutant. The results demonstrate that Wzx proteins from O-antigen systems that use N-acetylglucosamine or N-acetylgalactosamine for the initiation of the biosynthesis of the O repeat can fully complement the formation of O16 LPS. Partial complementation was seen with Wzx from Pseudomonas aeruginosa, a system that uses N-acetylfucosamine in the initiation reaction. In contrast, there was negligible complementation with the Wzx protein from Salmonella enterica, a system in which galactose is the initiating sugar. These results support a model whereby the first sugar of the O repeat can be recognized by the O-antigen translocation machinery.

Post-Translational Inhibition of IP-10 Secretion in IEC by Probiotic Bacteria: Impact on Chronic Inflammation

Background: Clinical and experimental studies suggest that the probiotic mixture VSL#3 has protective activities in the context of inflammatory bowel disease (IBD). The aim of the study was to reveal bacterial strain-specific molecular mechanisms underlying the anti-inflammatory potential of VSL#3 in intestinal epithelial cells (IEC).

Methodology/Principal Findings: VSL#3 inhibited TNF-induced secretion of the T-cell chemokine interferon-inducible protein (IP-10) in Mode-K cells. Lactobacillus casei (L. casei) cell surface proteins were identified as active anti-inflammatory components of VSL#3. Interestingly, L. casei failed to block TNF-induced IP-10 promoter activity or IP-10 gene transcription at the mRNA expression level but completely inhibited IP-10 protein secretion as well as IP-10-mediated T-cell transmigration. Kinetic studies, pulse-chase experiments and the use of a pharmacological inhibitor for the export machinery (brefeldin A) showed that L. casei did not impair initial IP-10 production but decreased intracellular IP-10 protein stability as a result of blocked IP-10 secretion. Although L. casei induced IP-10 ubiquitination, the inhibition of proteasomal or lysosomal degradation did not prevent the loss of intracellular IP-10. Most important for the mechanistic understanding, the inhibition of vesicular trafficking by 3-methyladenine (3-MA) inhibited IP-10 but not IL-6 expression, mimicking the inhibitory effects of L. casei. These findings suggest that L. casei impairs vesicular pathways important for the secretion of IP-10, followed by subsequent degradation of the proinflammatory chemokine. Feeding studies in TNF Delta ARE and IL-10(-/-) mice revealed a compartimentalized protection of VSL#3 on the development of cecal but not on ileal or colonic inflammation. Consistent with reduced tissue pathology in IL-10(-/-) mice, IP-10 protein expression was reduced in primary epithelial cells.

Conclusions/Significance: We demonstrate segment specific effects of probiotic intervention that correlate with reduced IP-10 protein expression in the native epithelium. Furthermore, we revealed post-translational degradation of IP-10 protein in IEC to be the molecular mechanism underlying the anti-inflammatory effect.

Nontypeable Haemophilus influenzae clearance by alveolar macrophages is impaired by exposure to cigarette smoke

Nontypeable Haemophilus influenzae (NTHI) is an opportunistic gram-negative pathogen that causes respiratory infections and is associated with progression of respiratory diseases. Cigarette smoke is a main risk factor for development of respiratory infections and chronic respiratory diseases. Glucocorticoids, which are anti-inflammatory drugs, are still the most common therapy for these diseases. Alveolar macrophages are professional phagocytes that reside in the lung and are responsible for clearing infections by the action of their phagolysosomal machinery and promotion of local inflammation. In this study, we dissected the interaction between NTHI and alveolar macrophages and the effect of cigarette smoke on this interaction. We showed that alveolar macrophages clear NTHI infections by adhesion, phagocytosis, and phagolysosomal processing of the pathogen. Bacterial uptake requires host actin polymerization, the integrity of plasma membrane lipid rafts, and activation of the phosphatidylinositol 3-kinase (PI3K) signaling cascade. Parallel to bacterial clearance, macrophages secrete tumor necrosis factor alpha (TNF-alpha) upon NTHI infection. In contrast, exposure to cigarette smoke extract (CSE) impaired alveolar macrophage phagocytosis, although NTHI-induced TNF-alpha secretion was not abrogated. Mechanistically, our data showed that CSE reduced PI3K signaling activation triggered by NTHI. Treatment of CSE-exposed cells with the glucocorticoid dexamethasone reduced the amount of TNF-alpha secreted upon NTHI infection but did not compensate for CSE-dependent phagocytic impairment. The deleterious effect of cigarette smoke was observed in macrophage cell lines and in human alveolar macrophages obtained from smokers and from patients with chronic obstructive pulmonary disease.

HIV-1 Nef limits communication between linker of activated T cells and SLP-76 to reduce formation of SLP-76-signaling microclusters following TCR stimulation

Signal initiation by engagement of the TCR triggers actin rearrangements, receptor clustering, and dynamic organization of signaling complexes to elicit and sustain downstream signaling. Nef, a pathogenicity factor of HIV, disrupts early TCR signaling in target T cells. To define the mechanism underlying this Nef-mediated signal disruption, we employed quantitative single-cell microscopy following surface-mediated TCR stimulation that allows for dynamic visualization of distinct signaling complexes as microclusters (MCs). Despite marked inhibition of actin remodeling and cell spreading, the induction of MCs containing TCR-CD3 or ZAP70 was not affected significantly by Nef. However, Nef potently inhibited the subsequent formation of MCs positive for the signaling adaptor Src homology-2 domain-containing leukocyte protein of 76 kDa (SLP-76) to reduce MC density in Nef-expressing and HIV-1-infected T cells. Further analyses suggested that Nef prevents formation of SLP-76 MCs at the level of the upstream adaptor protein, linker of activated T cells (LAT), that couples ZAP70 to SLP-76. Nef did not disrupt pre-existing MCs positive for LAT. However, the presence of the viral protein prevented de novo recruitment of active LAT into MCs due to retargeting of LAT to an intracellular compartment. These modulations in MC formation and composition depended on Nef's ability to simultaneously disrupt both actin remodeling and subcellular localization of TCR-proximal machinery. Nef thus employs a dual mechanism to disturb early TCR signaling by limiting the communication between LAT and SLP-76 and preventing the dynamic formation of SLP-76-signaling MCs.

Aspasing Out Metacaspases and Caspases: Proteases of Many Trades

Execution of programmed cell death (PCD) in nonmetazoan organisms is morphologically different from apoptotic PCD in animals and lacks a number of key molecular components of apoptotic machinery, including caspases. Yet protozoan, fungal, and plant cells exhibit caspase-like proteolytic activities, which increase in a PCD-dependent manner. This poses a question whether nonmetazoan organisms contain structurally dissimilar proteases that functionally substitute for caspases. Putative ancestors of caspases, metacaspases, are candidates for this role; however, their distinct substrate specificity raises doubts. The identification of a common biological target of caspases and metacaspases and previously unknown functions unrelated to cell death of metacaspases provide new food for thought.