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.     

Alpha-tocopheryl acetate is absorbed and hydrolyzed by Caco-2 cells comparative studies with alpha-tocopherol

Caco-2 cells were used as a model for investigating and comparing the absorption of alpha-tocopherol (Tol) and alpha-tocopheryl acetate (Tac) solubilized in micelles based on a mixture of sodium taurocholate (NaTC) and oleic acid. Surprisingly, the uptake of Tac was found to be similar to that of Tol, and in both cases, the dose-response plots suggest that protein-mediated transport processes were involved. Moreover Tol or Tac were also secreted into the basolateral medium of Caco-2 cells but Tac was mainly hydrolyzed either prior to absorption or intracellularly. The solubilization of Tol or Tac by NaTC on the apical side of the cell monolayer is a prerequisite for the uptake process, although larger amounts of the bile salt are necessary to solubilize Tac than Tol. Caco-2 cells showed hydrolytic activity on Tac, and additional cholesterol esterase may be taken up by the cells, thus increasing the rates of intracellular hydrolysis of Tac. Based on our findings, a scheme is suggested accounting for the absorption of alpha-tocopheryl acetate by enterocytes.     

Expression of subunits of the 19S complex and of the PA28 activator in rat skeletal muscle

A precise knowledge of the role of subunits of the 19S complex and the PA28 regulator, which associate with the 20S proteasome and regulate its peptidase activities, may contribute to design new therapeutic approaches for preventing muscle wasting in human diseases. The proteasome is mainly responsible for the muscle wasting of tumor-bearing and unweighted rats. The expression of some ATPase (MSS1, P45) and non ATPase (P112-L, P31) subunits of the 19S complex, and of the two subunits of the PA28 regulator, was studied in such atrophying muscles. The mRNA levels for all studied subunits increased in unweighted rats, and analysis of MSS1 mRNA distribution profile in polyribosomes showed that this subunit entered active translation. By contrast, only the mRNA levels for MSS1 increased in the muscles from cancer rats. Thus, gene expression of the proteasome regulatory subunits depends on a given catabolic state. Torbafylline, a xanthine derivative which inhibits tumor necrosis factor production, prevented the activation of protein breakdown and the increased expression of 20S proteasome subunits in cancer rats, without reducing the elevated MSS1 mRNA levels. Thus, the increased expression of MSS1 is regulated independently of 20S proteasome subunits, and did not result in accelerated proteolysis.     

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     

Lasioglossins LLIII affect the morphogenesis of Candida albicans and reduces the duration of experimental vaginal candidiasis in mice

Lasioglossins are a group of peptides with identified antimicrobial activity. The inhibitory effects of two synthetic lasioglossin derivatives, LLIII and D‐isomeric variant LLIII‐D, on morphological changes in Candida albicans in vitro and the effect of local administration of LLIII during experimental murine candidiasis were investigated. C. albicans blastoconidia were grown in the presence of lasioglossin LLIII or LLIII‐D at concentrations of 11.5 μM and 21 μM, respectively, for 1, 2 and 3 days and their viability determined by flow cytometry using eosin Y staining. Morphological changes were examined by light and fluorescent microscopy. The Candida‐inhibitory effect of daily intravaginal administration of 0.7 or 1.4 μg of LLIII was assessed in mice with experimentally‐induced vaginal candidiasis. LLIII and LLIII‐D lasioglossins exhibited candidacidal activity in vitro (>76% after 24 hr and >84% after 48 hr of incubation). After 72 hr incubation of Candida with low concentration of lasioglossins, an increase in viability was detected, probably due to a Candida antimicrobial peptides evasion strategy. Furthermore, lasioglossins inhibited temperature‐induced morphotype changes toward hyphae and pseudohyphae with sporadic occurrence of atypical cells with two or enlarged nuclei, suggesting interference with mitosis or cytokinesis. Local application of LLIII reduced the duration of experimental candidiasis with no evidence of adverse effects. Lasioglossin LLIII is a promising candidate for development as an antimicrobial drug for treating the vaginal candidiasis.