Localization of a 64-kDa phosphoprotein in the lumen between the outer and inner envelopes of pea chloroplasts

The identification and localization of a marker protein for the intermembrane space between the outer and inner chloroplast envelopes is described. This 64-kDa protein is very rapidly labeled by [γ-32P]ATP at very low (30 nM) ATP concentrations and the phosphoryl group exhibits a high turnover rate. It was possible to establish the presence of the 64-kDa protein in this plastid compartment by using different chloroplast envelope separation and isolation techniques. In addition comparison of labeling kinetics by intact and hypotonically lysed pea chloroplasts support the localization of the 64-kDa protein in the intermembrane space. The 64-kDa protein was present and could be labeled in mixed envelope membranes isolated from hypotonically lysed plastids. Mixed envelope membranes incorporated high amounts of 32P from [γ-32P]ATP into the 64-kDa protein, whereas separated outer and inner envelope membranes did not show significant phosphorylation of this protein. Water/Triton X-114 phase partitioning demonstrated that the 64-kDa protein is a hydrophilic polypeptide. These findings suggest that the 64-kDa protein is a soluble protein trapped in the space between the inner and outer envelope membranes. After sonication of mixed envelope membranes, the 64-kDa protein was no longer present in the membrane fraction, but could be found in the supernatant after a 110000 × g centrifugation

3D time series analysis of cell shape using Laplacian approaches

Background: Fundamental cellular processes such as cell movement, division or food uptake critically depend on cells being able to change shape. Fast acquisition of three-dimensional image time series has now become possible, but we lack efficient tools for analysing shape deformations in order to understand the real three-dimensional nature of shape changes. Results: We present a framework for 3D+time cell shape analysis. The main contribution is three-fold: First, we develop a fast, automatic random walker method for cell segmentation. Second, a novel topology fixing method is proposed to fix segmented binary volumes without spherical topology. Third, we show that algorithms used for each individual step of the analysis pipeline (cell segmentation, topology fixing, spherical parameterization, and shape representation) are closely related to the Laplacian operator. The framework is applied to the shape analysis of neutrophil cells. Conclusions: The method we propose for cell segmentation is faster than the traditional random walker method or the level set method, and performs better on 3D time-series of neutrophil cells, which are comparatively noisy as stacks have to be acquired fast enough to account for cell motion. Our method for topology fixing outperforms the tools provided by SPHARM-MAT and SPHARM-PDM in terms of their successful fixing rates. The different tasks in the presented pipeline for 3D+time shape analysis of cells can be solved using Laplacian approaches, opening the possibility of eventually combining individual steps in order to speed up computations

Identification and codon reading properties of 5-cyanomethyl uridine, a new modified nucleoside found in the anticodon wobble position of mutant haloarchaeal isoleucine tRNAs

Most archaea and bacteria use a modified C in the anticodon wobble position of isoleucine tRNA to base pair with A but not with G of the mRNA. This allows the tRNA to read the isoleucine codon AUA without also reading the methionine codon AUG. To understand why a modified C, and not U or modified U, is used to base pair with A, we mutated the C34 in the anticodon of Haloarcula marismortui isoleucine tRNA (tRNA2Ile) to U, expressed the mutant tRNA in Haloferax volcanii, and purified and analyzed the tRNA. Ribosome binding experiments show that although the wild-type tRNA2Ile binds exclusively to the isoleucine codon AUA, the mutant tRNA binds not only to AUA but also to AUU, another isoleucine codon, and to AUG, a methionine codon. The G34 to U mutant in the anticodon of another H. marismortui isoleucine tRNA species showed similar codon binding properties. Binding of the mutant tRNA to AUG could lead to misreading of the AUG codon and insertion of isoleucine in place of methionine. This result would explain why most archaea and bacteria do not normally use U or a modified U in the anticodon wobble position of isoleucine tRNA for reading the codon AUA. Biochemical and mass spectrometric analyses of the mutant tRNAs have led to the discovery of a new modified nucleoside, 5-cyanomethyl U in the anticodon wobble position of the mutant tRNAs. 5-Cyanomethyl U is present in total tRNAs from euryarchaea but not in crenarchaea, eubacteria, or eukaryotes.National Institutes of Health (U.S.) (GM17151)National Institutes of Health (U.S.) (GM22854)National Institutes of Health (U.S.) (ES017010)Singapore-MIT Alliance for Research and TechnologySingapore. National Research FoundationUnited States. Dept. of Energy (DE-FG36-08GO88055

Dialectics and difference: against Harvey's dialectical post-Marxism

David Harvey`s recent book, Justice, nature and the geography of difference (JNGD), engages with a central philosophical debate that continues to dominate human geography: the tension between the radical Marxist project of recent decades and the apparently disempowering relativism and `play of difference' of postmodern thought. In this book, Harvey continues to argue for a revised `post-Marxist' approach in human geography which remains based on Hegelian-Marxian principles of dialectical thought. This article develops a critique of that stance, drawing on the work of Jacques Derrida, Gilles Deleuze and Felix Guattari. I argue that dialectical thinking, as well as Harvey's version of `post-Marxism', has been undermined by the wide-ranging `post-' critique. I suggest that Harvey has failed to appreciate the full force of this critique and the implications it has for `post-Marxist' ontology and epistemology. I argue that `post-Marxism', along with much contemporary human geography, is constrained by an inflexible ontology which excessively prioritizes space in the theory produced, and which implements inflexible concepts. Instead, using the insights of several `post-' writers, I contend there is a need to develop an ontology of `context' leading to the production of `contextual theories'. Such theories utilize flexible concepts in a multilayered understanding of ontology and epistemology. I compare how an approach which produces a `contextual theory' might lead to more politically empowering theory than `post-Marxism' with reference to one of Harvey's case studies in JNGD

Phenotypic Variation and Bistable Switching in Bacteria

Microbial research generally focuses on clonal populations. However, bacterial cells with identical genotypes frequently display different phenotypes under identical conditions. This microbial cell individuality is receiving increasing attention in the literature because of its impact on cellular differentiation, survival under selective conditions, and the interaction of pathogens with their hosts. It is becoming clear that stochasticity in gene expression in conjunction with the architecture of the gene network that underlies the cellular processes can generate phenotypic variation. An important regulatory mechanism is the so-called positive feedback, in which a system reinforces its own response, for instance by stimulating the production of an activator. Bistability is an interesting and relevant phenomenon, in which two distinct subpopulations of cells showing discrete levels of gene expression coexist in a single culture. In this chapter, we address techniques and approaches used to establish phenotypic variation, and relate three well-characterized examples of bistability to the molecular mechanisms that govern these processes, with a focus on positive feedback.

Electroacupuncture versus Diclofenac in symptomatic treatment of Osteoarthritis of the knee: a randomized controlled trial

BACKGROUND: The purpose of this study was to compare the efficacy of electroacupuncture (EA), diclofenac and their combination in symptomatic treatment of osteoarthritis (OA) of the knee. METHODS: This study was a randomized, single-blind, placebo controlled trial. The 193 out-patients with OA of the knee were randomized into four groups: placebo, diclofenac, EA and combined (diclofenac plus EA). Paracetamol tablets were prescribed as a rescue analgesic during the study. The patients were evaluated after a run-in period of one week (week 0) and again at the end of the study (week 4). The clinical assessments included the amount of paracetamol taken/week, visual analog scale (VAS), Western Ontario and McMaster Universities (WOMAC) OA Index, Lequesne's functional index, 50 feet-walk time, and the orthopedist's and patient's opinion of change. RESULTS: One hundred and eighty six patients completed the study. The improvement of symptoms (reduction in mean changes) in most outcome parameters was greatest in the EA group. The proportions of responders and patients with an overall opinion of "much better" were also greatest in the EA group. The improvement in VAS was significantly different between the EA and placebo group as well as the EA and diclofenac group. The improvement in Lequesne's functional index also differed significantly between the EA and placebo group. In addition, there was a significant improvement in WOMAC pain index between the combined and placebo group. CONCLUSION: EA is significantly more effective than placebo and diclofenac in the symptomatic treatment of OA of the knee in some circumstances. However, the combination of EA and diclofenac treatment was no more effective than EA treatment alone

Belief Revision and Delusions: How Do Patients with Schizophrenia Take Advice?

The dominant cognitive model that accounts for the persistence of delusional beliefs in schizophrenia postulates that patients suffer from a general deficit in belief revision. It is generally assumed that this deficit is a consequence of impaired reasoning skills. However, the possibility that such inflexibility affects the entire system of a patient's beliefs has rarely been empirically tested. Using delusion-neutral material in a well-documented advice-taking task, the present study reports that patients with schizophrenia: 1) revise their beliefs, 2) take into account socially provided information to do so, 3) are not overconfident about their judgments, and 4) show less egocentric advice-discounting than controls. This study thus shows that delusional patients' difficulty in revising beliefs is more selective than had been previously assumed. The specificities of the task and the implications for a theory of delusion formation are discussed