In vitro characterization of a plastid terminal oxidase (PTOX).

The plastid terminal oxidase (PTOX) encoded by the Arabidopsis IMMUTANS gene was expressed in Escherichia coli cells and its quinone/oxygen oxidoreductase activity monitored in isolated bacterial membranes using NADH as an electron donor. Specificity for plastoquinone was observed. Neither ubiquinone, duroquinone, phylloquinone nor benzoquinone could substitute for plastoquinone in this assay. However, duroquinol (fully reduced chemically) was an accepted substrate. Iron is also required and cannot be substituted by Cu(2+), Zn(2+) or Mn(2+). This plastoquinol oxidase activity is independent of temperature over the 15-40 degrees C range but increases with pH (from 5.5 to 9.0). Unlike higher plant mitochondrial alternative oxidases, to which PTOX shows sequence similarity (but also differences, especially in a putative quinone binding site and in cysteine conservation), PTOX activity does not appear to be regulated by pyruvate or any other tested sugar, nor by AMP. Its activity decreases, however, with increasing salt (NaCl or KCl) concentration. Various quinone analogues were tested for their inhibitory activity on PTOX. Pyrogallol analogues were found to be inhibitors, especially octyl gallate (I50 = 0.4 microM ) that appears far more potent than propyl gallate or gallic acid. Thus, octyl gallate is a useful inhibitor for future in vivo or in organello studies aimed at studying the roles of PTOX in chlororespiration and as a cofactor for carotenoid biosynthesis.     

Zooplankton influence on the phytoplankton-bacterioplankton coupling

The evolution of phytoplankton biomass, and bacterial numberand activity were followed in laboratory microcosms with andwithout zooplankton, in order to study the time lag betweenphytoplankton and bacterioplankton development, as well as thepossible influence of consumers on phytoplankton-bacterioplanktoncoupling.     

Calmodulin and calcium differentially regulate the neuronal Nav1.1 voltage-dependent sodium channel

Mutations in the neuronal Nav1.1 voltage-gated sodium channel are responsible for mild to severe epileptic syndromes. The ubiquitous calcium sensor calmodulin (CaM) bound to rat brain Nav1.1 and to the human Nav1.1 channel expressed by a stably transfected HEK-293 cell line. The C-terminal region of the channel, as a fusion protein or in the yeast two-hybrid system, interacted with CaM via a consensus C-terminal motif, the IQ domain. Patch clamp experiments on HEK1.1 cells showed that CaM overexpression increased peak current in a calcium-dependent way. CaM had no effect on the voltage-dependence of fast inactivation, and accelerated the inactivation kinetics. Elevating Ca⁺⁺ depolarized the voltage-dependence of fast inactivation and slowed down the fast inactivation kinetics, and for high concentrations this effect competed with the acceleration induced by CaM alone. Similarly, the depolarizing action of calcium antagonized the hyperpolarizing shift of the voltage-dependence of activation due to CaM overexpression. Fluorescence spectroscopy measurements suggested that Ca⁺⁺ could bind the Nav1.1 C-terminal region with micromolar affinity.     

Valid and reliable instruments for arm-hand assessment at ICF activity level in persons with hemiplegia: a systematic review

Background

Loss of arm-hand performance due to a hemiparesis as a result of stroke or cerebral palsy (CP), leads to large problems in daily life of these patients. Assessment of arm-hand performance is important in both clinical practice and research. To gain more insight in e.g. effectiveness of common therapies for different patient populations with similar clinical characteristics, consensus regarding the choice and use of outcome measures is paramount. To guide this choice, an overview of available instruments is necessary. The aim of this systematic review is to identify, evaluate and categorize instruments, reported to be valid and reliable, assessing arm-hand performance at the ICF activity level in patients with stroke or cerebral palsy.

Methods

A systematic literature search was performed to identify articles containing instruments assessing arm-hand skilled performance in patients with stroke or cerebral palsy. Instruments were identified and divided into the categories capacity, perceived performance and actual performance. A second search was performed to obtain information on their content and psychometrics.

Results

Regarding capacity, perceived performance and actual performance, 18, 9 and 3 instruments were included respectively. Only 3 of all included instruments were used and tested in both patient populations. The content of the instruments differed widely regarding the ICF levels measured, assessment of the amount of use versus the quality of use, the inclusion of unimanual and/or bimanual tasks and the inclusion of basic and/or extended tasks.

Conclusions

Although many instruments assess capacity and perceived performance, a dearth exists of instruments assessing actual performance. In addition, instruments appropriate for more than one patient population are sparse. For actual performance, new instruments have to be developed, with specific focus on the usability in different patient populations and the assessment of quality of use as well as amount of use. Also, consensus about the choice and use of instruments within and across populations is needed.     

Heme Oxygenase-1 Regulates the Progression of K/BxN Serum Transfer Arthritis

Background

Heme oxygenase-1 (HO-1) is induced in many cell types as a defense mechanism against stress. We have investigated the possible role of endogenous HO-1 in the effector phase of arthritis using the K/BxN serum transfer model of arthritis in HO-1 heterozygous and homozygous knock-out mice.

Methodology/Principal Findings

Arthritis was induced in C57/Black-6 xFVB (HO-1^+/+, HO-1^+/− and HO-1^−/−) mice by intraperitoneal injection of 150 µl serum from arthritic K/BxN mice at days 0 and 2. Blood was collected and animals were sacrificed at day 10. Histological analysis was performed in ankle sections. The levels of inflammatory mediators were measured in serum and paw homogenates by enzyme-linked immunosorbent assay or Multiplex technology. The incidence of arthritis was higher in HO-1^+/− and HO-1^−/− groups compared with HO-1^+/+. The inflammatory response was aggravated in HO-1^+/− mice as shown by arthritic score and the migration of inflammatory cells that could be related to the enhancement of CXCL-1 production. In addition, the HO-1^+/− group showed proteoglycan depletion significantly higher than HO-1^+/+ mice. Serum levels of matrix metalloproteinase-3, monocyte chemotactic protein-1, plasminogen activator inhibitor-1, E-selectin and intercellular adhesion molecule-1 were increased in arthritic HO-1^−/− mice, whereas vascular endothelial growth factor and some cytokines such as interferon-γ showed a reduction compared to HO-1^+/+ or HO-1^+/− mice. In addition, down-regulated gene expression of ferritin, glutathione S-reductase A1 and superoxide dismutase-2 was observed in the livers of arthritic HO-1^+/− animals.

Conclusion/Significance

Endogenous HO-1 regulates the production of systemic and local inflammatory mediators and plays a protective role in K/BxN serum transfer arthritis.     

Biological interactions in enclosed plankton communities including Alexandrium catenella and copepods: Role of phosphorus

A microcosm approach was used to test whether: a) growth under unbalanced nutrient conditions (varying N:P ratios) affected the susceptibility of a phytoplankton community including the dinoflagellate Alexandrium catenella (a paralytic shellfish toxin producer) to mesozooplankton grazing, and b) the potential effects of unbalanced nutrient conditions were mediated by changes in toxicity of A. catenella or by other mechanisms. The experimental setup consisted of fifteen 30 l microcosms, filled with water from the Barcelona Harbour and subjected to treatments combining nutrient inputs at three different N:P ratios (Redfield N:P ratio or nutrient-balanced, high N:P and low N:P), addition or omission of A. catenella (an estimated initial concentration of 38 A. catenella cells ml^− 1, a value typical for blooms in harbours of the Catalan coast), and selective addition of a cultured population of Acartia grani. P sufficiency had a strong positive effect on the growth of A. grani, both with or without A. catenella addition, presumably due to enhanced food quality of the prey community. The presence of this copepod resulted in lower concentrations of ciliates, A. catenella, and other dinoflagellates, suggesting active grazing by the copepods. No noxious effects of A. catenella on the copepods were detected at the relatively low cell concentrations of that dinoflagellate used in the experiment.     

Diffusion, Exclusion, and Specific Binding in a Large Channel: A Study of OmpF Selectivity Inversion

We find that moderate cationic selectivity of the general bacterial porin OmpF in sodium and potassium chloride solutions is inversed to anionic selectivity in concentrated solutions of barium, calcium, nickel, and magnesium chlorides. To understand the origin of this phenomenon, we consider several factors, which include the binding of divalent cations, electrostatic and steric exclusion of differently charged and differently sized ions, size-dependent hydrodynamic hindrance, electrokinetic effects, and significant “anionic” diffusion potential for bulk solutions of chlorides of divalent cations. Though all these factors contribute to the measured selectivity of this large channel, the observed selectivity inversion is mostly due to the following two. First, binding divalent cations compensates, or even slightly overcompensates, for the negative charge of the OmpF protein, which is known to be the main cause of cationic selectivity in sodium and potassium chloride solutions. Second, the higher anionic (versus cationic) transport rate expected for bulk solutions of chloride salts of divalent cations is the leading cause of the measured anionic selectivity of the channel. Interestingly, at high concentrations the binding of cations does not show any pronounced specificity within the divalent series because the reversal potentials measured in the series correlate well with the corresponding bulk diffusion potentials. Thus our study shows that, in contrast to the highly selective channels of neurophysiology that employ mostly the exclusion mechanism, quite different factors account for the selectivity of large channels. The elucidation of these factors is essential for understanding large channel selectivity and its regulation in vivo.