The stoichiometry of the chloroplast ATP synthase oligomer III in Chlamydomonas reinhardtii is not affected by the metabolic state

The chloroplast H(+)-ATP synthase is a key component for the energy supply of higher plants and green algae. An oligomer of identical protein subunits III is responsible for the conversion of an electrochemical proton gradient into rotational motion. It is highly controversial if the oligomer III stoichiometry is affected by the metabolic state of any organism. Here, the intact oligomer III of the ATP synthase from Chlamydomonas reinhardtii has been isolated for the first time. Due to the importance of the subunit III stoichiometry for energy conversion, a gradient gel system was established to distinguish oligomers with different stoichiometries. With this methodology, a possible alterability of the stoichiometry in respect to the metabolic state of the cells was examined. Several growth parameters, i.e., light intensity, pH value, carbon source, and CO(2) concentration, were varied to determine their effects on the stoichiometry. Contrary to previous suggestions for E. coli, the oligomer III of the chloroplast H(+)-ATP synthase always consists of a constant number of monomers over a wide range of metabolic states. Furthermore, mass spectrometry indicates that subunit III from C. reinhardtii is not modified posttranslationally. Data suggest a subunit III stoichiometry of the algae ATP synthase divergent from higher plants.     

Mechanisms regulating the positioning of mouse p47 resistance GTPases LRG-47 and IIGP1 on cellular membranes: retargeting to plasma membrane induced by phagocytosis

The recently identified p47 GTPases are one of the most effective cell-autonomous resistance systems known against intracellular pathogens in the mouse. One member of the family, LRG-47, has been shown to be essential for immune control in vivo of Listeria monocytogenes, Toxoplasma gondii, Mycobacterium tuberculosis, and Mycobacterium avium, possibly by promoting acidification of the phagosome. However, the intracellular localization of LRG-47, and the nature of its association with the phagosomal or any other membrane system is unknown. In this study, we show that LRG-47 is a Golgi-associated protein in the IFN-stimulated cell, which is rapidly recruited to active plasma membrane upon phagocytosis and remains associated with phagosomes as they mature. We show that the Golgi localization of LRG-47 is dependent on the integrity of an amphipathic helix near the C terminus, whereas the plasma membrane localization depends on an unidentified signal associated with the G domain. Unlike LRG-47, but like the published p47 resistance GTPase, IGTP, a further p47 GTPase, IIGP1, is associated with the endoplasmic reticulum. However, unlike IGTP, IIGP1 is associated with the endoplasmic reticulum by an N-terminal myristoylation modification. Thus, the p47 GTPases are a diverse battery of intracellular defense factors dynamically associated with different membrane systems.     

The SPA quartet: a family of WD-repeat proteins with a central role in suppression of photomorphogenesis in arabidopsis

The Arabidopsis thaliana proteins suppressor of phytochrome A-105 1 (SPA1), SPA3, and SPA4 of the four-member SPA1 protein family have been shown to repress photomorphogenesis in light-grown seedlings. Here, we demonstrate that spa quadruple mutant seedlings with defects in SPA1, SPA2, SPA3, and SPA4 undergo strong constitutive photomorphogenesis in the dark. Consistent with this finding, adult spa quadruple mutants are extremely small and dwarfed. These extreme phenotypes are only observed when all SPA genes are mutated, indicating functional redundancy among SPA genes. Differential contributions of individual SPA genes were revealed by analysis of spa double and triple mutant genotypes. SPA1 and SPA2 predominate in dark-grown seedlings, whereas SPA3 and SPA4 prevalently regulate the elongation growth in adult plants. Further analysis of SPA2 function indicated that SPA2 is a potent repressor of photomorphogenesis only in the dark but not in the light. The SPA2 protein is constitutively nuclear localized in planta and can physically interact with the repressor COP1. Epistasis analysis between spa2 and cop1 mutations provides strong genetic support for a biological significance of a COP1-SPA2 interaction in the plant. Taken together, our results have identified a new family of proteins that is essential for suppression of photomorphogenesis in darkness.     

Signaling through a Novel Domain of gp130 Mediates Cell Proliferation and Activation of Hck and Erk Kinases

Interleukin-6 (IL-6) induces the activation of the Src family kinase Hck, which is associated with the IL-6 receptor beta-chain, gp130. Here we describe the identification of an "acidic" domain comprising amino acids 771 to 811 of gp130 as a binding region for Hck, which mediates proliferative signaling. The deletion of this region of gp130 (i.e., in deletion mutant d771-811) resulted in a significant reduction of Hck kinase activity and cell proliferation upon stimulation of gp130 compared to wild-type gp130. In addition, d771-811 disrupted the growth factor-stimulated activation of Erk and the dephosphorylation of Pyk2. Based on these findings, we propose a novel, acidic domain of gp130, which is responsible for the activation of Hck, Erk, and Pyk2 and signals cell proliferation upon growth factor stimulation.     

Na+/K+-ATPase inhibition during cardiac myocyte swelling: Involvement of intracellular pH and Ca2+

Previous studies in chick embryo cardiac myocytes have shown that the inhibition of Na⁺/K⁺-ATPase with ouabain induces cell shrinkage in an isosmotic environment (290 mOsm). The same inhibition produces an enhanced RVD (regulatory volume decrease) in hyposmotic conditions (100 mOsm). It is also known that submitting chick embryo cardiomyocytes to a hyperosmotic solution induces shrinkage and a concurrent intracellular alkalization. The objective of this study was to evaluate the involvement of intracellular pH (pH_i), intracellular Ca²⁺ ([Ca²⁺]_i) and Na⁺/K⁺-ATPase inhibition during hyposmotic swelling. Changes in intracellular pH and Ca²⁺ were monitored using BCECF and fura-2, respectively. The addition of ouabain (100 M) under both isosmotic and hyposmotic stimuli resulted in a large increase in [Ca²⁺]_i (200%). A decrease in pH_i (from 7.3 ± 0.09 to 6.4 ± 0.08, n = 6; p < 0.05) was only observed when ouabain was applied during hyposmotic swelling. This acidification was prevented by the removal of extracellular Ca²⁺. Inhibition of Na⁺/H²⁺ exchange with amiloride (1 mM) had no effect on the ouabain-induced acidification. Preventing the mitochondrial accumulation of Ca²⁺ using CCCP (10 M) resulted in a blockade of the progressive acidification normally induced by ouabain. The inhibition of mitochondrial membrane K⁺/H⁺ exchange with DCCD (1 mM) also completely prevented the acidification. Our results suggest that intracellular acidification upon cell swelling is mediated by an initial Ca²⁺ influx via Na⁺/Ca²⁺ exchange, which under hyposmotic conditions activates the K⁺ and Ca²⁺ mitochondrial exchange systems (K⁺/H⁺ and Ca²⁺/H⁺).Deceased     

Synergistic effects of mixed TiAlV and polyethylene wear particles on TNFalpha response in THP-1 macrophages.

TNFalpha is a potent osteoclastogenic cytokine that has a fundamental role in the pathogenesis of wear particle-induced osteolysis. Wear particles of one composition and their biological effects are well characterised. In contrast, little is known about the effects of mixed particles with respect to mix ratio and particle concentration. We evaluated the effects of different mix ratios of polyethylene and TiAlV particles on TNFalpha response. We used a human monocytic cell line (THP-1) in this in vitro study. THP-1 monocytes were differentiated to macrophage-like cells and exposed to different mixtures of lipopolysaccharide-detoxified polyethylene and TiAlV particles. TNFalpha was analysed in culture supernatants using ELISAs. Both polyethylene and TiAlV particles induced a dose- and time-related release of TNFalpha, with maximum levels after 6 h. A PE/TiAlV mix ratio of 36:1 at 10(8) particles/ml induced significantly higher TNFalpha concentrations compared to equal particle concentrations of isolated TiAlV (p=0.047) or PE (p=0.044), indicating the synergistic effect of mixed particles. These results provide evidence that TiAlV and polyethylene particles have significant synergistic effects, depending on the mix ratio and particle concentrations. This supra-additive effect can contribute substantially to the pathogenesis of implant particle-induced osteolysis.     

Regulation of aggregate size and pattern by adenosine and caffeine in cellular slime molds

Background

Multicellularity in cellular slime molds is achieved by aggregation of several hundreds to thousands of cells. In the model slime mold Dictyostelium discoideum, adenosine is known to increase the aggregate size and its antagonist caffeine reduces the aggregate size. However, it is not clear if the actions of adenosine and caffeine are evolutionarily conserved among other slime molds known to use structurally unrelated chemoattractants. We have examined how the known factors affecting aggregate size are modulated by adenosine and caffeine.

Result

Adenosine and caffeine induced the formation of large and small aggregates respectively, in evolutionarily distinct slime molds known to use diverse chemoattractants for their aggregation. Due to its genetic tractability, we chose D. discoideum to further investigate the factors affecting aggregate size. The changes in aggregate size are caused by the effect of the compounds on several parameters such as cell number and size, cell-cell adhesion, cAMP signal relay and cell counting mechanisms. While some of the effects of these two compounds are opposite to each other, interestingly, both compounds increase the intracellular glucose level and strengthen cell-cell adhesion. These compounds also inhibit the synthesis of cAMP phosphodiesterase (PdsA), weakening the relay of extracellular cAMP signal. Adenosine as well as caffeine rescue mutants impaired in stream formation (pde4 ^- and pdiA ^- ) and colony size (smlA ^- and ctnA ^- ) and restore their parental aggregate size.

Conclusion

Adenosine increased the cell division timings thereby making large number of cells available for aggregation and also it marginally increased the cell size contributing to large aggregate size. Reduced cell division rates and decreased cell size in the presence of caffeine makes the aggregates smaller than controls. Both the compounds altered the speed of the chemotactic amoebae causing a variation in aggregate size. Our data strongly suggests that cytosolic glucose and extracellular cAMP levels are the other major determinants regulating aggregate size and pattern. Importantly, the aggregation process is conserved among different lineages of cellular slime molds despite using unrelated signalling molecules for aggregation.     

Biomimetically mineralized salmon collagen scaffolds for application in bone tissue engineering

Biomimetic mineralization of collagen is an advantageous method to obtain resorbable collagen/hydroxy-apatite composites for application in bone regeneration. In this report, established procedures for mineralization of bovine collagen were adapted to a new promising source of collagen from salmon skin challenged by the low denaturation temperature. Therefore, in the first instance, variation of temperature, collagen concentration, and ionic strength was performed to reveal optimized parameters for fibrillation and simultaneous mineralization of salmon collagen. Porous scaffolds from mineralized salmon collagen were prepared by controlled freeze-drying and chemical cross-linking. FT-IR analysis demonstrated the mineral phase formed during the preparation process to be hydroxyapatite. The scaffolds exhibited interconnecting porosity, were sufficiently stable under cyclic compression, and showed elastic mechanical properties. Human mesenchymal stem cells were able to adhere to the scaffolds, cell number increased during cultivation, and osteogenic differentiation was demonstrated in terms of alkaline phosphatase activity.