CML9, a multifunctional Arabidopsis thaliana calmodulin-like protein involved in stress responses and plant growth?

Plants have evolved complex signaling networks to respond to their fluctuating environment and adapt their growth and development. Calcium-dependent signaling pathways play key role in the onset of these adaptive responses. In plant cells, the intracellular calcium transients are triggered by numerous stimuli and it is supposed that the large repertory of calcium sensors present in higher plants could contribute to integrate these signals in physiological responses. Here, we present data on CML9, a calmodulin-like protein that appears to be involved in plant responses to both biotic and abiotic stress. Using a reverse genetic approach based on gain and loss of function mutants, we present here data indicating that this CML might also be involved in root growth control in response to the flagellin, a pathogen-associated molecular pattern (PAMP) also involved in plant immunity.     

BICARBONATE STIMULATION OF CALCIFICATION AND PHOTOSYNTHESIS IN TWO HERMATYPIC CORALS1

A wide range of bicarbonate concentrations was used to monitor the kinetics of bicarbonate (HCO₃^?) use in both photosynthesis and calcification in two reef‐building corals, Porites porites and Acropora sp. Experiments carried out close to the P. porites collection site in Barbados showed that additions of NaHCO₃ to synthetic seawater proportionally increased the calcification rate of this coral until the concentration exceeded three times that of seawater (6 mM). Photosynthetic rates were also stimulated by HCO₃^? addition, but these became saturated at a lower concentration (4 mM). Similar experiments on aquarium‐acclimated colonies of Indo‐Pacific Acropora sp. showed that calcification and photosynthesis in this coral were enhanced to an even greater extent than P. porites, with calcification continuing to increase above 8 mM HCO₃^?, and photosynthesis saturating at 6 mM. Calcification rates of Acropora sp. were also monitored in the dark, and, although these were lower than in the light for a given HCO₃^? concentration, they still increased dramatically with HCO₃^? addition, showing that calcification in this coral is light stimulated but not light dependent.     

The ubiquitin-proteasome and the mitochondria-associated apoptotic pathways are sequentially downregulated during recovery after immobilization-induced muscle atrophy

Immobilization produces morphological, physiological, and biochemical alterations in skeletal muscle leading to muscle atrophy and long periods of recovery. Muscle atrophy during disuse results from an imbalance between protein synthesis and proteolysis but also between apoptosis and regeneration processes. This work aimed to characterize the mechanisms underlying muscle atrophy and recovery following immobilization by studying the regulation of the mitochondria-associated apoptotic and the ubiquitin-proteasome-dependent proteolytic pathways. Animals were subjected to hindlimb immobilization for 4-8 days (I4 to I8) and allowed to recover after cast removal for 10-40 days (R10 to R40). Soleus and gastrocnemius muscles atrophied from I4 to I8 to a greater extent than extensor digitorum longus and tibialis anterior muscles. Gastrocnemius muscle atrophy was first stabilized at R10 before being progressively reduced until R40. Polyubiquitinated proteins accumulated from I4, whereas the increased ubiquitination rates and chymotrypsin-like activity of the proteasome were detectable from I6 to I8. Apoptosome and caspase-3 or -9 activities increased at I6 and I8, respectively. The ubiquitin-proteasome-dependent pathway was normalized early when muscle stops to atrophy (R10). By contrast, the mitochondria-associated apoptotic pathway was first downregulated below basal levels when muscle started to recover at R15 and completely normalized at R20. Myf 5 protein levels decreased from I4 to I8 and were normalized at R10. Altogether, our results suggest a two-stage process in which the ubiquitin-proteasome pathway is rapidly up- and downregulated when muscle atrophies and recovers, respectively, whereas apoptotic processes may be involved in the late stages of atrophy and recovery.     

4Pi microscopy reveals an impaired three-dimensional mitochondrial network of pancreatic islet β-cells,an experimental model of type-2 diabetes

Insulin production in pancreatic β-cells is critically linked to mitochondrial oxidative phosphorylation. Increased ATP production triggered by blood glucose represents the β-cells' glucose sensor. Type-2 diabetes mellitus results from insulin resistance in peripheral tissues and impaired insulin secretion. Pathology of diabetic β-cells might be reflected by the altered morphology of mitochondrial network. Its characterization is however hampered by the complexity and density of the three-dimensional (3D) mitochondrial tubular networks in these cell types. Conventional confocal microscopy does not provide sufficient axial resolution to reveal the required details; electron tomography reconstruction of these dense networks is still difficult and time consuming. However, mitochondrial network morphology in fixed cells can also be studied by 4Pi microscopy, a laser scanning microscopy technique which provides an ～ 7-fold improved axial resolution (～ 100 nm) over conventional confocal microscopy. Here we present a quantitative study of these networks in insulinoma INS-1E cells and primary β-cells in Langerhans islets. The former were a stably-transfected cell line while the latter were transfected with lentivirus, both expressing mitochondrial matrix targeted redox-sensitive GFP. The mitochondrial networks and their partial disintegration and fragmentation are revealed by carefully created iso-surface plots and their quantitative analysis. We demonstrate that β-cells within the Langerhans islets from diabetic Goto Kakizaki rats exhibited a more disintegrated mitochondrial network compared to those from control Wistar rats and model insulinoma INS-1E cells. Standardization of these patterns may lead to development of morphological diagnostics for Langerhans islets, for the assessment of β-cell condition, before their transplantations.     

Dinoflagellate cyst distribution in marine surface sediments off West Africa (17–6°N) in relation to sea-surface conditions, freshwater input and seasonal coastal upwelling

An organic-walled dinoflagellate cyst analysis was carried out on 53 surface sediment samples from West Africa (17–6°N) to obtain insight in the relationship between their spatial distribution and hydrological conditions in the upper water column as well as marine productivity in the study area.Multivariate analysis of the dinoflagellate cyst relative abundances and environmental parameters of the water column shows that sea-surface temperature, salinity, marine productivity and bottom water oxygen are the factors that relate significantly to the distribution patterns of individual species in the region.The composition of cyst assemblages and dinoflagellate cyst concentrations allows the identification of four hydrographic regimes; 1) the northern regime between 17 and 14°N characterized by high productivity associated with seasonal coastal upwelling, 2) the southern regime between 12 and 6°N associated with high-nutrient waters influenced by river discharge 3) the intermediate regime between 14 and 12°N influenced mainly by seasonal coastal upwelling additionally associated with fluvial input of terrestrial nutrients and 4) the offshore regime characterized by low chlorophyll-a concentrations in upper waters and high bottom water oxygen concentrations.Our data show that cysts of Polykrikos kofoidii, Selenopemphix quanta, Dubridinium spp., Echinidinium species, cysts of Protoperidinium monospinum and Spiniferites pachydermus are the best proxies to reconstruct the boundary between the NE trade winds and the monsoon winds in the subtropical eastern Atlantic Ocean. The association of Bitectatodinium spongium, Lejeunecysta oliva, Quinquecuspis concreta, Selenopemphix nephroides, Trinovantedinium applanatum can be used to reconstruct past river outflow variations within this region.     

Biotransformation of 4-halophenols to 4-halocatechols using Escherichia coli expressing 4-hydroxyphenylacetate 3-hydroxylase

Escherichia coli cells, expressing 4-hydroxyphenylacetate 3-hydroxylase, fully transformed 4-halogenated phenols to their equivalent catechols as single products in shaken flasks. 4-Fluorophenol was transformed at a rate 1.6, 1.8, and 3.4-fold higher than the biotransformation of 4-chloro-, 4-bromo-, and 4-iodo-phenol, respectively. A scale-up from shaken flask to a 5 L stirred tank bioreactor was undertaken to develop a bioprocess for the production of 4-substituted halocatechols at higher concentrations and scale. In a stirred tank reactor, the optimized conditions for induction of 4-HPA hydroxylase expression were at 37 °C for 3 h. The rate of biotransformation of 4-fluorophenol to 4-fluorocatechol by stirred tank bioreactor grown cells was the same at 1 and 4.8 mM (5.13 μmol/min/g CDW) once the ratio of biocatalyst (E. coli CDW) to substrate concentration (mM) was maintained at 2:1. At 10.8 mM 4-fluorophenol, the rate of 4-fluorocatechol formation decreased by 4.7-fold. However, the complete transformation of 1.3 g of 4-fluorophenol (10.8 mM) to 4-fluorocatechol was achieved within 7 h in a 1 L reaction volume. Similar to 4-fluorophenol, other 4-substituted halophenols were completely transformed to 4-halocatechols at 2 mM within a 1–2 h period. An increase in 4-halophenol concentration to 4.8 mM resulted in a 2.5–20-fold decrease in biotransformation efficiency depending on the substrate tested. Organic solvent extraction of the 4-halocatechol products followed by column chromatography resulted in the production of purified products with a final yield of between 33% and 38%.     

Performance of ¿VIKIA Malaria Ag Pf/Pan¿ (IMACCESS®), a new malaria rapid diagnostic test for detection of symptomatic malaria infections

ABSTRACT: BACKGROUND: Recently, IMACCESS[REGISTERED SIGN] developed a new malaria test (VIKIA Malaria Ag Pf/Pan[TRADE MARK SIGN]), based on the detection of falciparum malaria (HRP-2) and non-falciparum malaria (aldolase). METHODS: The performance of this new malaria rapid diagnostic test (RDT) was assessed using 1,000 febrile patients seeking malaria treatment in four health centres in Cambodia from August to December 2011. The results of the VIKIA Malaria Ag Pf/Pan were compared with those obtained by microscopy, the CareStart Malaria[TRADE MARK SIGN] RDT (AccessBio[REGISTERED SIGN]) which is currently used in Cambodia, and real-time PCR (as "gold standard"). RESULTS: The best performances of the VIKIA Malaria Ag Pf/Pan[TRADE MARK SIGN] test for detection of both Plasmodium falciparum and non-P. falciparum were with 20--30 min reading times (sensitivity of 93.4% for P. falciparum and 82.8% for non-P. falciparum and specificity of 98.6% for P. falciparum and 98.9% for non-P. falciparum) and were similar to those for the CareStart Malaria[TRADE MARK SIGN] test. CONCLUSIONS: This new RDT performs similarly well as other commercially available tests (especially the CareStart Malaria[TRADE MARK SIGN] test, used as comparator), and conforms to the World Health Organization's recommendations for RDT performance. It is a good alternative tool for the diagnosis of malaria in endemic areas.     

Endocytosis by Numb breaks Notch symmetry at cytokinesis

Cell-fate diversity can be generated by the unequal segregation of the Notch regulator Numb at mitosis in both vertebrates and invertebrates. Whereas the mechanisms underlying unequal inheritance of Numb are understood, how Numb antagonizes Notch has remained unsolved. Live imaging of Notch in sensory organ precursor cells revealed that nuclear Notch is detected at cytokinesis in the daughter cell that does not inherit Numb. Numb and Sanpodo act together to regulate Notch trafficking and establish directional Notch signalling at cytokinesis. We propose that unequal segregation of Numb results in increased endocytosis in one daughter cell, hence asymmetry of Notch at the cytokinetic furrow, directional signalling and binary fate choice.     

Curcumin treatment prevents increased proteasome and apoptosome activities in rat skeletal muscle during reloading and improves subsequent recovery

Immobilization is characterized by activation of the ubiquitin (Ub)-proteasome-dependent proteolytic system (UPS) and of the mitochondrial apoptotic pathway. Increased oxidative stress and inflammatory response occur in immobilized skeletal muscles. Curcumin exhibits antioxidant and anti-inflammatory properties, blocked proteasome activation in intact animals, and may favor skeletal muscle regeneration. We therefore measured the effects of curcumin on immobilization-induced muscle atrophy and subsequent recovery. Rats were subjected to hindlimb immobilization for 8 days (I8) and allowed to recover for 10 days (R10). Fifty percent of the rats were injected daily with either curcumin or vehicle. Proteolytic and apoptotic pathways were studied in gastrocnemius muscles. Curcumin treatment prevented the enhanced proteasome chymotrypsin-like activity and the trend toward increased caspase-9-associated apoptosome activity at I8 in immobilized muscles. By contrast, the increase of these two activities was blunted by curcumin at R10. Curcumin did not reduce muscle atrophy at I8 but improved muscle recovery at R10 and the cross-sectional area of muscle fibers of immobilized muscles. Curcumin reduced the increased protein levels of Smac/DIABLO induced by immobilization and enhanced the elevation of X-linked inhibitory apoptotic protein levels at R10. Ub-conjugate levels and caspase-3 activity increased at I8 and were normalized at R10 without being affected by curcumin treatment. Altogether, the data show that curcumin treatment improved recovery during reloading. The effect of curcumin during the atrophic phase on proteasome activities may facilitate the initiation of muscle recovery after reloading. These data also suggest that this compound may favor the initial steps of muscle regeneration.     

Interactions of DNA with a new electron-deficient tentacle porphyrin: meso-Tetrakis[2,3,5,6-tetrafluoro-4-(2-trimethylammoniumethylamine)phenyl]porphyrin

A new electron-deficient tentacle porphyrin meso-tetrakis[2,3,5,6-tetrafluoro-4-(2-trimethylammoniumethylamine)phenyl]porphyrin (TθF₄TAP) has been synthesized. The binding interactions of TθF₄TAP with DNA polymers were studied for comparison to those of an electron-deficient tentacle porphyrin and an electron-rich tentacle porphyrin; these previously studied porphyrins bind to DNA primarily by intercalative and outside-binding modes, respectively. The three tentacle porphyrins have similar size and shape. The basicity of TθF₄TAP indicated that it has electronic characteristics similar to those of the intercalating electron-deficient tentacle porphyrin. However, TθF₄TAP binds to calf thymus DNA, [poly(dA-dT)]₂, and [poly(dG-dC)]₂ in a self-stacking, outside-binding manner under all conditions. Evidence for this binding mode included a significant hypochromicity of the Soret band, a conservative induced CD spectrum, and the absence of an increase in DNA solution viscosity. As found previously for the electron-rich porphyrin, the results suggest that combinations of closely related self-stacked forms coexist. The mix of forms depended on the DNA and the solution conditions. There are probably differences in the detailed features of the self-stacking adducts for the two types of tentacle porphyrins, especially at high R (ratio of porphyrin to DNA). At low R values, the induced CD signal of TθF₄TAP/CT DNA resembled that of TθF₄TAP/[poly(dA-dT)]₂, suggesting that TθF₄TAP binds preferentially at AT regions. Competitive binding experiments gave evidence that TθF₄TAP binds preferentially to [poly(dA-dT)]₂ over [poly(dG-dC)]₂. Thus, despite the long, positively charged, flexible substituents on the porphyrin, the binding of TθF₄TAP is significantly affected by base-pair composition. Similar characteristics were found previously for the electron-rich tentacle porphyrin. Thus, significant changes in electron richness have relatively minor effects on this outside binding selectivity for AT regions. TθF₄TAP is the first porphyrin with electron deficiency and shape similar to intercalating porphyrins that does not appear to intercalate. All porphyrins reported to intercalate have had pyridinium substituents. Thus, the electronic distribution in the porphyrin ring, not just the overall electron richness, may play a role in facilitating intercalation. © 1997 John Wiley & Sons, Inc. Biopoly 42: 203–217, 1997