The Arabidopsis COP9 signalosome is essential for G2 phase progression and genomic stability

The COP9 signalosome (CSN) is required for the full activity of cullin-RING E3 ubiquitin ligases (CRLs) in eukaryotes. CSN exerts its function on CRLs by removing the ubiquitin-related NEDD8 conjugate from the cullin subunit of CRLs. CSN seems, thereby, to control CRL disassembly or CRL subunit stability. In Arabidopsis thaliana, loss of CSN function leads to constitutive photomorphogenic (cop) seedling development and a post-germination growth arrest. The underlying molecular cause of this growth arrest is currently unknown. Here, we show that Arabidopsis csn mutants are delayed in G2 phase progression. This cell cycle arrest correlates with the induction of the DNA damage response pathway and is suggestive of the activation of a DNA damage checkpoint. In support of this hypothesis, we detected gene conversion events in csn mutants that are indicative of DNA double-strand breaks. DNA damage is also apparent in mutants of the NEDD8 conjugation pathway and in mutants of the E3 ligase subunits CULLIN4, COP1 and DET1, which share phenotypes with csn mutants. In summary, our data suggest that Arabidopsis csn mutants undergo DNA damage, which might be the cause of the delay in G2 cell cycle progression.     

Low over-expression of TNFalpha in the mouse heart increases contractile performance via TNFR1

TNFalpha is a cytokine wit pleiotropic functions in many organs. In the heart increased TNFalpha levels are not only associated with heart failure, but also, paradoxically, with protection from ischemic damage. To test whether the protective role of TNFalpha in the heart is concentration-dependent, we studied two mouse heart models with low (two- to threefold) over-expression of endogenous TNFalpha: mice deficient in a translational repressor of TNFalpha mRNA, TIA-1(-/-), and mice over-expressing human TNFalpha. Hearts lacking TIA-1 were characterized for their endogenous TNFalpha over-expression during normal Langendorff perfusion. To define which TNFalpha receptor mediates cardiac protection, we also used mice lacking the TNFR1 receptor. Contractile function was assessed in isolated hearts perfused in the isovolumic Langendorff mode during and following global no-flow ischemic stress and in response to varying extracellular [Ca(2+)] to determine their contractile response and Ca(2+) sensitivity. All hearts with low over-expression of TNFalpha, independent of human or murine origin, have improved contractile performance and increased Ca(2+) sensitivity (by 0.2-0.26 pCa). Hearts lacking TNFR1 have contractile performance equal to wild type hearts. Recovery from ischemia was greater in TIA-1(-/-) and was diminished in TNFR1(-/-). Better contractile function in TNFalpha over-expressing hearts is not due to improved cardiac energetics assessed as [ATP] and glucose uptake or to differences in expression of SERCA2a or calmodulin. We suggest that low levels of TNFalpha increase the Ca(2+) sensitivity of the heart via a TNFR1-mediated mechanism.     

The relocation of starch metabolism to chloroplasts: when, why and how

Plastid endosymbiosis was accompanied by the appearance of a novel type of semi-cristalline storage polysaccharide (starch). Interestingly, starch is found in the cytoplasm of Rhodophyceae and Glaucophyta but is localized to the chloroplast stroma of Chloroplastida. The pathway is presumed to have been cytosolic in the common ancestor of the three Archaeplastida lineages. The means by which in green plants and algae an entire suite of nuclear-encoded starch-metabolism genes could have had their protein products rewired simultaneously to plastids are unclear. This opinion article reviews the timing and the possible reasons underlying this rewiring and proposes a hypothesis that explains its mechanism. The consequences of this mechanism on the complexity of starch metabolism in Chloroplastida are discussed.     

Nonmitochondrial ATP/ADP Transporters Accept Phosphate as Third Substrate

Chlamydiales and Rickettsiales as metabolically impaired, intracellular pathogenic bacteria essentially rely on “energy parasitism” by the help of nucleotide transporters (NTTs). Also in plant plastids NTT-type carriers catalyze ATP/ADP exchange to fuel metabolic processes. The uptake of ATP^4-, followed by energy consumption and the release of ADP^3-, would lead to a metabolically disadvantageous accumulation of negative charges in form of inorganic phosphate (P_i) in the bacterium or organelle if no interacting P_i export system exists. We identified that P_i is a third substrate of several NTT-type ATP/ADP transporters. During adenine nucleotide hetero-exchange, P_i is cotransported with ADP in a one-to-one stoichiometry. Additionally, P_i can be transported in exchange with solely P_i. This P_i homo-exchange depends on the presence of ADP and provides a first indication for only one binding center involved in import and export. Furthermore, analyses of mutant proteins revealed that P_i interacts with the same amino acid residue as the γ-phosphate of ATP. Import of ATP in exchange with ADP plus P_i is obviously an efficient way to couple energy provision with the export of the two metabolic products (ADP plus P_i) and to maintain cellular phosphate homeostasis in intracellular living “energy parasites” and plant plastids. The additional P_i transport capacity of NTT-type ATP/ADP transporters makes the existence of an interacting P_i exporter dispensable and might explain why a corresponding protein so far has not been identified.Most organisms possess the capacity to resynthesize the fundamental energy currency ATP by fusion of ADP and P_i. Generally, in eukaryotes the major part of energy is produced in specialized organelles, the mitochondria. Mitochondrial ADP/ATP carriers (AACs)² mediate the export of newly synthesized ATP in strict counter-exchange with cytosolic ADP and therefore provide energy to the cellular metabolism (1). Plants additionally generate high amounts of ATP during photosynthesis in chloroplasts. However, under conditions of limiting or missing photosynthetic activity, plant plastids depend on external energy supply (2–4). Specific nucleotide transporters (NTTs) located in the inner plastid envelope membrane mediate the required energy import (5). These transporters structurally, functionally, and phylogenetically differ from mitochondrial AACs. They catalyze the import of cytosolic ATP in exchange with stromal ADP, are monomers consisting of 12 predicted transmembrane helices, and are related to the functionally heterogeneous group of bacterial NTTs (5).Although most prokaryotic organisms are able to regenerate ATP and therefore are considered as energetically self-sustaining, the obligate intracellular living bacterial orders Chlamydiales and Rickettsiales are impaired in energy and nucleotide synthesis or even completely lost the corresponding pathways (6–8). Therefore, these bacteria, which comprise important human pathogens (9, 10), essentially rely on nucleotide and energy import. Bacterial NTTs catalyze the required import of a broad range of nucleotides and NAD or facilitate the counter-exchange of ATP and ADP (5, 11–15). The latter process has been termed “energy parasitism” and obviously is of high importance for the survival of rickettsial and chlamydial cells (5, 16–18).Although import measurements on intact Escherichia coli cells expressing the corresponding proteins allowed characterization of many bacterial and plastidial NTTs (12–15, 19–24), a very important physiological question is still not clarified. The uptake of ATP^4- in exchange with ADP^3- in absence of a concerted P_i export would result in a charge difference and a phosphate imbalance in the bacterial cell. In mitochondria, phosphate carriers metabolically cooperate with AACs because they provide P_i for ATP synthesis (25). Similarly, it was assumed that NTT-type ATP/ADP transporters cooperate with phosphate exporters to guarantee phosphate homeostasis in the bacterium or plastid. However, a P_i exporter interacting with ATP/ADP transporters is not known in “energy parasites” or plant plastids. Bacterial and plant phosphate transport systems rather facilitate P_i import or the counter-exchange of P_i and phosphorylated compounds and therefore do not allow net P_i export (26–29). Furthermore, the newly identified plastidial (proton-driven) phosphate transporters are not preferentially expressed under conditions or in tissues that require ATP provision to the plastid (30, 31).Recently, we succeeded in the purification of the first recombinant NTT from Protochlamydia amoebophila (PamNTT1), a parachlamydial endosymbiont of the protist Acantamoeba (32). The functional reconstitution of the highly pure PamNTT1 into artificial lipid vesicles for the first time allowed the biochemical characterization of a representative nonmitochondrial ATP/ADP transporter unaffected by the complex metabolic situation of the bacterial cell. We demonstrated that in contrast to mitochondrial AACs, PamNTT1 catalyzes a membrane potential independent, electroneutral adenine nucleotide hetero-exchange (32, 33). The latter could argue for a cotransport of a counterion compensating for the electrogenic ATP^4-/ADP^3- exchange.Here, we investigated possible ions accompanying ATP or ADP transport. Interestingly, we uncovered that PamNTT1 and also rickettsial and plastidial ATP/ADP transporters accept an additional important substrate, which is P_i. We performed a comprehensive characterization of the P_i transport and gained new insights into the transport properties of ATP/ADP transporters.     

A brilliant monomeric red fluorescent protein to visualize cytoskeleton dynamics in Dictyostelium

Red fluorescent proteins (RFPs) combined with GFP are attractive probes for double-fluorescence labeling of proteins in live cells. However, the application of these proteins is restrained by stable oligomer formation and by their weak fluorescence in vivo. Previous attempts to eliminate these problems by mutagenesis of RFP from Discosoma (DsRed) resulted in the monomeric mRFP1 and in the tetrameric RedStar RFP, which is distinguished by its enhanced fluorescence in vivo. Based on these mutations, we have generated an enhanced monomeric RFP, mRFPmars, and report its spectral properties. Together with green fluorescent labels, we used mRFPmars to visualize filamentous actin structures and microtubules in Dictyostelium cells. This enhanced RFP proved to be suitable to monitor the dynamics of cytoskeletal proteins in cell motility, mitosis, and endocytosis using dual-wavelength fluorescence microscopy.     

A new type of thermoluminometer: a highly sensitive tool in applied photosynthesis research and plant stress physiology

Here we describe a newly developed thermoluminescence measuring device that employs flash excitation, peltier heating, and light detection by channel photomultipliers (CPM). The new thermoluminometer is equipped with four sample holders for simultaneous measurements of thermoinduced light emission in the temperature range from -20 degrees C to +180 degrees C. It allows one to measure leaf samples, chloroplasts, thylakoids, algae, or even bioorganic material lacking chlorophyll by means of naturally induced or artificially applied chemilumigenic probes. The temperature range of the thermoluminometer allows one to analyse the thermoinduced radical pair recombination of photosystem II in the lower temperature region as well as chemiluminescence from lipid peroxidation in the higher temperature region. Hence, plant material can be assessed concerning both its photosynthetic and its oxidative stress status. Since the device is equipped with four sample holders and four CPM channels for simultaneous detection of thermoinduced light emission, it facilitates a high throughput. Therefore, the new device is interesting, not only in ecophysiology, but also in the field of plant breeding, as it can be used to study the stress tolerance of various cultivars of cultural crop plants.     

Enzyme catalysis via control of activation entropy: site-directed mutagenesis of 6,7-dimethyl-8-ribityllumazine synthase

6,7-Dimethyl-8-ribityllumazine synthase (lumazine synthase) catalyses the penultimate step in the biosynthesis of riboflavin. In Bacillus subtilis, 60 lumazine synthase subunits form an icosahedral capsid enclosing a homotrimeric riboflavin synthase unit. The ribH gene specifying the lumazine synthase subunit can be expressed in high yield. All amino acid residues exposed at the surface of the active site cavity were modified by PCR assisted mutagenesis. Polar amino acid residues in direct contact with the enzyme substrates, 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and 3,4-dihydroxy-2-butanone 4-phosphate, could be replaced with relative impunity with regard to the catalytic properties. Only the replacement of Arg127, which forms a salt bridge with the phosphate group of 3,4-dihydroxy-2-butanone 4-phosphate, reduced the catalytic rate by more than one order of magnitude. Replacement of His88, which is believed to assist in proton transfer reactions, reduced the catalytic activity by about one order of magnitude. Surprisingly, the activation enthalpy deltaH of the lumazine synthase reaction exceeds that of the uncatalysed reaction. On the other hand, the free energy of activation deltaG of the uncatalysed reaction is characterised by a large entropic term (TdeltaS) of -37.8 kJmol(-1), whereas the entropy of activation (TdeltaS) of the enzyme-catalysed reaction is -6.7 kJmol(-1). This suggests that the rate enhancement by the enzyme is predominantly achieved by establishing a favourable topological relation of the two substrates, whereas acid/base catalysis may play a secondary role.     

Nitrogen limitation of growth and nutrient dynamics in a disturbed mangrove forest,Indian River Lagoon,Florida

The objectives of this study were to determine effects of nutrient enrichment on plant growth, nutrient dynamics, and photosynthesis in a disturbed mangrove forest in an abandoned mosquito impoundment in Florida. Impounding altered the hydrology and soil chemistry of the site. In 1997, we established a factorial experiment along a tree-height gradient with three zones, i.e., fringe, transition, dwarf, and three fertilizer treatment levels, i.e., nitrogen (N), phosphorus (P), control, in Mosquito Impoundment 23 on the eastern side of Indian River. Transects traversed the forest perpendicular to the shoreline, from a Rhizophora mangle-dominated fringe through an Avicennia germinans stand of intermediate height, and into a scrub or dwarf stand of A. germinans in the hinterland. Growth rates increased significantly in response to N fertilization. Our growth data indicated that this site is N-limited along the tree-height gradient. After 2 years of N addition, dwarf trees resembled vigorously growing saplings. Addition of N also affected internal dynamics of N and P and caused increases in rates of photosynthesis. These findings contrast with results for a R. mangle-dominated forest in Belize where the fringe is N-limited, but the dwarf zone is P-limited and the transition zone is co-limited by N and P. This study demonstrated that patterns of nutrient limitation in mangrove ecosystems are complex, that not all processes respond similarly to the same nutrient, and that similar habitats are not limited by the same nutrient when different mangrove forests are compared.     

No influence of magnetic fields on cell cycle progression using conditions relevant for patients during MRI

The purpose of this study was to examine whether exposure to magnetic fields (MFs) relevant for magnetic resonance imaging (MRI) in clinical routine influences cell cycle progression in two tumor cell lines in vitro. HL60 and EA2 cells were exposed to four types of MFs: (i) static MF of 1.5 and 7.05 T, (ii) extremely low frequency magnetic gradient fields (ELFMGFs) with +/- 10 mT/m and 100 Hz, as well as +/- 100 mT/m and 100 Hz, (iii) pulsed high frequency MF in the radiofrequency (RF) range (63.6 MHz, 5.8 microT), and (iv) a combination of (i-iii). Exposure periods ranged from 1 to 24 h. Cell cycle distribution (G(0)/G(1), S, and G(2)/M phases) was analyzed by flow cytometry. Cell cycle analysis did not reveal differences between the exposed and the control cells. As expected, positive controls with irradiated (8 Gy) HL60 and EA2 cells showed a strong G(2)/M arrest. Using conditions that are relevant for patients during MRI, no influence of MFs on cell cycle progression was observed in these cell lines. Care was taken to control secondary parameters of influence, such as vibration by the MR scanner or temperature to avoid false positive results.