Forces,failles et opportunités de la recherche française en écologie trophique

The French National Institute of Ecology and Environment (INEE) aims at fostering pluridisciplinarity in Environmental Science and, for that purpose, funds ex muros research groups (GDR) on thematic topics. Trophic ecology has been identified as a scientific field in ecology that would greatly benefit from such networking activity, as being profoundly scattered. This has motivated the seeding of a GDR, entitled “GRET”. The contours of the GRET's action, and its ability to fill these gaps within trophic ecology at the French national scale, will depend on the causes of this relative scattering. This study relied on a nationally broadcasted poll aiming at characterizing the field of trophic ecology in France. Amongst all the unique individuals that fulfilled the poll, over 300 belonged at least partly to the field of trophic ecology. The sample included all French public research institutes and career stages. Three main disruptions within the community of scientist in trophic ecology were identified. The first highlighted the lack of interfaces between microbial and trophic ecology. The second evidenced that research questions were strongly linked to single study fields or ecosystem type. Last, research activities are still quite restricted to the ecosystem boundaries. All three rupture points limit the conceptual and applied progression in the field of trophic ecology. Here we show that most of the disruptions within French Trophic Ecology are culturally inherited, rather than motivated by scientific reasons or justified by socio-economic stakes. Comparison with the current literature confirms that these disruptions are not necessarily typical of the French research landscape, but instead echo the general weaknesses of the international research in ecology. Thereby, communication and networking actions within and toward the community of trophic ecologists, as planned within the GRET's objectives, should contribute to fill these gaps, by reintegrating microbes within trophic concepts and setting the seeds for trans- and meta-ecosystemic research opportunities. Once the community of trophic ecologists is aware of the scientific benefit in pushing its boundaries forwards, turning words and good intentions into concrete research projects will depend on the opportunities to obtain research funding.     

A Protein Related to Eucaryal and Bacterial DNA-Motor Proteins in the Hyperthermophilic Archaeon Sulfolobus acidocaldarius

We have isolated a new gene encoding a putative 103-kDa protein from the hyperthermophilic archaeon Sulfolobus acidocaldarius. Analysis of the deduced amino-acid sequence shows an extended central domain, predicted to form coiled-coil structures, and two terminal domains that display purine NTPase motifs. These features are reminiscent of mechanochemical motor proteins which use the energy of ATP hydrolysis to move specific cellular components. Comparative analysis of the amino-acid sequence of the terminal domains and predicted structural organization of this putative purine NTPase show that it is related both to eucaryal proteins from the ``SMC family' involved in the condensation of chromosomes and to several bacterial and eucaryal proteins involved in DNA recombination/repair. Further analyses revealed that these proteins are all members of the so called ``UvrA-related NTP-binding proteins superfamily' and form a large subgroup of motor-like NTPases involved in different DNA processing mechanisms. The presence of such protein in Archaea, Bacteria, and Eucarya suggests an early origin of DNA-motor proteins that could have emerged and diversified by domain shuffling. Received: 29 June 1996 / Accepted: 28 February 1997     

Crystal structure of the coat protein of the flexible filamentous papaya mosaic virus

The terminase motors of bacteriophages have been shown to be among the strongest active machines in the biomolecular world, being able to package several tens of kilobase pairs of viral genome into a capsid within minutes. Yet, these motors are hindered at the end of the packaging process by the progressive buildup of a force-resisting packaging associated with already packaged DNA. In this experimental work, we raise the issue of what sets the upper limit on the length of the genome that can be packaged by the terminase motor of phage λ and still yield infectious virions and the conditions under which this can be efficiently performed. Using a packaging strategy developed in our laboratory of building phage λ from scratch, together with plaque assay monitoring, we have been able to show that the terminase motor of phage λ is able to produce infectious particles with up to 110% of the wild-type λ-DNA length. However, the phage production rate, and thus the infectivity, decreased exponentially with increasing DNA length and was a factor of 10(3) lower for the 110% λ-DNA phage. Interestingly, our in vitro strategy was still efficient in fully packaging phages with DNA lengths as high as 114% of the wild-type length, but these viruses were unable to infect bacterial cells efficiently. Further, we demonstrated that the phage production rate is modulated by the presence of multivalent ionic species. The biological consequences of these findings are discussed.     

Autoprocessing of the Escherichia coli AIDA-I Autotransporter: A NEW MECHANISM INVOLVING ACIDIC RESIDUES IN THE JUNCTION REGION*

The cleavage of the autotransporter adhesin involved in diffuse adherence (AIDA-I) of Escherichia coli yields a membrane-embedded fragment, AIDAc, and an extracellular fragment, the mature AIDA-I adhesin. The latter remains noncovalently associated with AIDAc but can be released by heat treatment. In this study we determined the mechanism of AIDA-I cleavage. We showed that AIDA-I processing is an autocatalytic event by monitoring the in vitro cleavage of an uncleaved mutant protein isolated from inclusion bodies. Furthermore, by following changes in circular dichroism spectra and protease resistance of the renaturated protein, we showed that the cleavage of the protein is correlated with folding. With site-directed deletions, we showed that the catalytic activity of the protein lies in a region encompassing amino acids between Ala-667 and Thr-953, which includes the conserved junction domain of some autotransporters. With site-directed point mutations, we also found that Asp-878 and Glu-897 are involved in the processing of AIDA-I and that a mutation preserving the acidic side chain of Asp-878 was tolerated, giving evidence that this carboxylic acid group is directly involved in catalysis. Last, we confirmed that cleavage of AIDA-I is intramolecular. Our results unveil a new mechanism of auto-processing in the autotransporter family.Monomeric autotransporters, secreted by the type Va secretion pathway, constitute one of the largest family of secreted proteins in Gram-negative bacteria (1). Various virulence attributes have been associated with most autotransporters, such as adhesion or invasion, self-association, biofilm formation, serum resistance, and cytotoxic activity to name just a few (2, 3). Autotransporters are synthesized as pre-proproteins with modular organizations. An N-terminal sec-dependent signal sequence permits the secretion of the protein across the inner membrane (4). A C-terminal membrane-embedded domain promotes secretion of parts of the protein across the outer membrane and is composed of a β-barrel and a α-helix spanning its lumen (5, 6). The central domain of the protein is, thus, extracellular and bears its functional part. In some cases the extracellular part of the protein is cleaved and remains associated with the outer membrane or is secreted in the extracellular milieu. Directly preceding the membrane-embedded domain, a functional subdomain of ∼100 amino acids is sometimes present in the extracellular domain and has been called the junction region (also named autochaperone domain or stable core) (7–9). This region is essential for stabilizing the β-barrel and/or to promote folding of the extracellular domain (7–9).The adhesin involved in diffuse adherence (AIDA-I)³ is a monomeric autotransporter that has been extensively studied. AIDA-I was originally identified as a plasmid-encoded protein from a diffusely adhering Escherichia coli strain isolated in a case of infantile diarrhea (10). This adhesin was then shown to play a role in neonatal and postweaning diarrheal diseases in piglets, both of which cause major economic losses in farms worldwide (11–13). Besides its role as an adhesin, AIDA-I has been shown to mediate self-association and biofilm formation (14) as well as invasion of epithelial cells (15). Additionally, this protein undergoes a modification that is rare in bacteria, as it is O-glycosylated by the specific cytoplasmic protein autotransporter adhesin heptosyltransferase (Aah) (16, 17). AIDA-I has been suggested to be a member of a new group of autotransporter called self-associating autotransporters, which includes the Ag43 aggregation factor and the TibA invasin (18).After secretion at the cell surface, the extracellular domain of the protein, called mature AIDA-I, is cleaved from the C-terminal domain, called AIDAc, which encompasses the β-barrel, the α-helix, and the junction region (19, 20). Mature AIDA-I, however, remains strongly associated with AIDAc (15). The processing of AIDA-I is neither essential for the multiple functions of the protein nor for its stability (15). Questions remain about the mechanism involved in this cleavage. In a few autotransporters, the cleavage reaction involves an exogenous protease. IcsA for instance, an autotransporter of Shigella flexneri, is cleaved by a dedicated outer membrane protease called IcsP, which is related to the OmpT protease found in E. coli (21). Similarly, the Neisseria meningitidis serine protease NalP is specifically responsible for the partial processing of various autotransporters, including the IgA protease (22), App (22), AusI (23), and MspA (24). In contrast, the cleavage of AIDA-I is not mediated by the known E. coli periplasmic or outer membrane proteases (DegP, OmpT, or OmpP), and it occurs in E. coli as well as in Shigella or Salmonella strains (19). Based on those observations, this processing has been suggested to be an autocatalytic event, even though no protease catalytic site can be identified in the protein.Two types of autocatalytic processing mechanisms have been described in other autotransporters. The Hap autotransporter of Haemophilus influenzae (25), the SphB1 protein of Bordetella pertussis (26), and the NalP (22), App (27), and IgA proteases of N. meningitidis (28) cleave themselves by an endogenous serine protease domain. More recently, it was shown that the serine protease autotransporters of Enterobacteriaceae (SPATE) family cleave themselves in the α-helix located in the lumen of the membrane-embedded β-barrel by a catalytic reaction that involves an aspartate and a conserved asparagine (6, 29). The same autocatalytic reaction has been shown for two distantly related B. pertussis autotransporters, pertactin and BrkA (29). However, none of these mechanisms can be hypothesized for AIDA-I cleavage; AIDA-I possesses neither a serine protease domain like Hap nor the asparagine residue required for the SPATE cleavage. In addition, the AIDA-I cleavage site is not located in the α-helix. Thus, the mechanism of AIDA-I processing must be different and remains unknown.The mechanisms by which a diversity of other autotransporters are cleaved remain unknown. This is for instance the case for the E. coli Ag43 self-associating autotransporters (30), the Helicobacter pylori VacA cytotoxin (31), the Chlamydia pneumoniae polymorphic membrane proteins (32), and the Ssp proteins of Serratia marcescens (33) as well as the cohemolysin Cfa (34) and the Arp immunogenic protein of Bartonella henselae (35).In this study we determined the mechanism of AIDA-I cleavage. We first showed that a truncated mutant of AIDA-I can refold in vitro and undergo self-cleavage, providing definite proof for the autocatalytic mechanism. By constructing site-directed deletions and point mutations, we then showed that the catalytic domain lies in the region encompassing the amino acids between Ala-667 and Thr-953, and we identified two residues in the junction region, Asp-878 and Glu-897, that are essential for processing. Last, we confirmed that the mechanism of proteolysis for AIDA-I is intramolecular. This is the first report of this type of autocatalytic activity.     

The Fifth Transmembrane Domain of Angiotensin II Type 1 Receptor Participates in the Formation of the Ligand-binding Pocket and Undergoes a Counterclockwise Rotation upon Receptor Activation

The octapeptide hormone angiotensin II exerts a wide variety of cardiovascular effects through the activation of the angiotensin II Type 1 (AT₁) receptor, which belongs to the G protein-coupled receptor superfamily. Like other G protein- coupled receptors, the AT₁ receptor possesses seven transmembrane domains that provide structural support for the formation of the ligand-binding pocket. The role of the fifth transmembrane domain (TMD5) was investigated using the substituted cysteine accessibility method. All of the residues within Thr-190 to Leu-217 region were mutated one at a time to cysteine, and after expression in COS-7 cells, the mutant receptors were treated with the sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA). MTSEA reacts selectively with water-accessible, free sulfhydryl groups of endogenous or introduced point mutation cysteines. If a cysteine is found in the binding pocket, the covalent modification will affect the binding kinetics of the ligand. MTSEA substantially decreased the binding affinity of L197C-AT₁, N200C-AT₁, I201C-AT₁, G203C-AT₁, and F204C-AT₁ mutant receptors, which suggests that these residues orient themselves within the water-accessible binding pocket of the AT₁ receptor. Interestingly, this pattern of acquired MTSEA sensitivity was altered for TMD5 reporter cysteines engineered in a constitutively active N111G-AT₁ receptor background. Indeed, mutant I201C-N111G-AT₁ became more sensitive to MTSEA, whereas mutant G203C-N111G-AT₁ lost some sensitivity. Our results suggest that constitutive activation of AT₁ receptor causes an apparent counterclockwise rotation of TMD5 as viewed from the extracellular side.     

Age-dependent oxidative stress: toward an irreversible failure in endothelial maintenance

In order to maintain cellular homeostasis against endogenous and exogenous aggressions, different cellular mechanisms of defence, maintenance and repair are continuously activated throughout life. Hormesis, a concept based on the fact that mild stresses protect cells against subsequent stresses, amplifies the efficacy of the cellular mechanisms of defence and repair. Ageing, senescence and ultimately death, result from the exhaustion of these mechanisms maintaining cellular functions. One of the major sources of vascular endothelial damage is oxidative stress. The age-dependent shift in the redox environment towards pro-oxidation contributes to a progressive compensatory remodelling of the endothelium, an accumulation of damages, and its dysfunction, the premises for atherosclerosis. We propose that in agreement with the concept of hormesis, a moderate exposure during endothelial maturation to mild physiological oxidative stressors determines -vascular longevity.     

Study on the role of calmodulin in sperm function through the enrichment and identification of calmodulin-binding proteins in bovine ejaculated spermatozoa

Calmodulin is a small, highly conserved acidic protein present at high levels in spermatozoa that mediates numerous intracellular Ca²⁺-dependent events. Sperm motility and fertilizing ability results from an array of biochemical pathways under Ca²⁺ control, in which the importance of calmodulin is not fully understood. The role of calmodulin in sperm function has been mostly assessed using antagonists. Nevertheless, few known calmodulin-regulated enzymes have been described in spermatozoa regarding their involvement in sperm function. To further understand the role of this important Ca²⁺ mediator in spermatozoa, different studies were also undertaken to investigate and to identify sperm calmodulin-binding proteins and determine their localization and subcellular distribution as an attempt to elucidate the role of this important Ca²⁺ mediator. In the present study, sperm calmodulin-binding proteins were identified by mass spectrometry after Ca²⁺-dependent biotinylated-calmodulin binding on sperm head proteins subjected to 2D electrophoresis and transferred on a polyvinylidene difluoride membrane. Calmodulin binding protein identification was also done on detergent extracted whole sperm proteins pulled down in a Ca²⁺-dependent manner by calmodulin-conjugated sepharose beads. In this latter group, 300 proteins were identified in at least two experiments out of three, and those identified in the three independent experiments were analyzed for overrepresented biological processes using the Bos taurus Gene Ontology database. Proteins with known function in reproductive processes, fertilization, sperm-egg recognition, sperm binding to the zona pellucida, regulation of sperm capacitation, and sperm motility were identified and further emphasize the importance of calmodulin in sperm function.     

Validation and Reliability of a Classification Method to Measure the Time Spent Performing Different Activities

The aim of this study was to validate the performance and reliability of results obtained from a classification model that measures time spent performing activities in confined (CE) and unrestricted (UE) environments. In CE, participants wore a pair of biaxial and/or triaxial accelerometers while performing pre-determined training activities classified as variants of lying down, dynamic standing, sitting, walking and running on two separate days. A classification model trained with activities performed in a specific order during the first day was developed to validate the activities performed in a random order on the second day (CE) and over 24 hours on a separate day (UE). The performance of the classification model was validated against triaxial accelerometers using six (x, y and step counts for arm and thigh) or eight (same as six features plus z axis) features. The reliability of the classification model was tested in both environments using six features. Results revealed an overall accuracy of 94% in CE and 90% in UE. The sensitivity in CE and UE was 94% and 95% for lying down, 88% and 80% for dynamic standing, 97% and 89% for sitting, 96% and 78% for walking and 90% and 64% for running, respectively. No significant differences were noted between performances obtained with six or eight features. Results were highly reproducible in both environments. The results obtained from the classification model were accurate and reproducible, and highlight the potential use of this approach in research to quantify the time spent performing different activities.     

Neural overlap in processing music and speech

Neural overlap in processing music and speech, as measured by the co-activation of brain regions in neuroimaging studies, may suggest that parts of the neural circuitries established for language may have been recycled during evolution for musicality, or vice versa that musicality served as a springboard for language emergence. Such a perspective has important implications for several topics of general interest besides evolutionary origins. For instance, neural overlap is an important premise for the possibility of music training to influence language acquisition and literacy. However, neural overlap in processing music and speech does not entail sharing neural circuitries. Neural separability between music and speech may occur in overlapping brain regions. In this paper, we review the evidence and outline the issues faced in interpreting such neural data, and argue that converging evidence from several methodologies is needed before neural overlap is taken as evidence of sharing.