Catabolic ornithine carbamoyltransferase of Pseudomonas aeruginosa. Importance of the N-terminal region for dodecameric structure and homotropic carbamoylphosphate cooperativity.

Pseudomonas aeruginosa has an anabolic (ArgF) and a catabolic (ArcB) ornithine carbamoyltransferase (OTCase). Despite extensive sequence similarities, these enzymes function unidirectionally in vivo. In the dodecameric catabolic OTCase, homotropic cooperativity for carbamoylphosphate strongly depresses the anabolic reaction; the residue Glu1O5 and the C-terminus are known to be essential for this cooperativity. When Glu1O5 and nine C-terminal amino acids of the catabolic OTCase were introduced, by in vitro genetic manipulation, into the closely related, trimeric, anabolic (ArgF) OTCase of Escherichia coli, the enzyme displayed Michaelis-Menten kinetics and no cooperativity was observed. This indicates that additional amino acid residues are required to produce homotropic cooperativity and a dodecameric assembly. To localize these residues, we constructed several hybrid enzymes by fusing, in vivo or in vitro, the E. coli argF gene to the P. aeruginosa arcB gene. A hybrid enzyme consisting of 101 N-terminal ArgF amino acids fused to 233 C-terminal ArcB residues and the reciprocal ArcB-ArgF hybrid were both trimers with little or no cooperativity. Replacing the seven N-terminal residues of the ArcB enzyme by the corresponding six residues of E. coli ArgF enzyme produced a dodecameric enzyme which showed a reduced affinity for carbamoylphosphate and an increase in homotropic cooperativity. Thus, the N-terminal amino acids of catabolic OTCase are important for interaction with carbamoylphosphate, but do not alone determine dodecameric assembly. Hybrid enzymes consisting of either 26 or 42 N-terminal ArgF amino acids and the corresponding C-terminal ArcB residues were both trimeric, yet they retained some homotropic cooperativity. Within the N-terminal ArcB region, a replacement of motif 28-33 by the corresponding ArgF segment destabilized the dodecameric structure and the enzyme existed in trimeric and dodecameric states, indicating that this region is important for dodecameric assembly. These findings were interpreted in the light of the three-dimensional structure of catabolic OTCase, which allows predictions about trimer-trimer interactions. Dodecameric assembly appears to require at least three regions: the N- and C-termini (which are close to each other in a monomer), residues 28-33 and residues 147-154. Dodecameric structure correlates with high carbamoylphosphate cooperativity and thermal stability, but some trimeric hybrid enzymes retain cooperativity, and the dodecameric Glu1O5-->Ala mutant gives hyperbolic carbamoylphosphate saturation, indicating that dodecameric structure is neither necessary nor sufficient to ensure cooperativity.

Short term evolution of a highly transmissible methicillin-resistant Staphylococcus aureus clone (ST228) in a tertiary care hospital.

Staphylococcus aureus is recognized as one of the major human pathogens and is by far one of the most common nosocomial organisms. The genetic basis for the emergence of highly epidemic strains remains mysterious. Studying the microevolution of the different clones of S. aureus is essential for identifying the forces driving pathogen emergence and spread. The aim of the present study was to determine the genetic changes characterizing a lineage belonging to the South German clone (ST228) that spread over ten years in a tertiary care hospital in Switzerland. For this reason, we compared the whole genome of eight isolates recovered between 2001 and 2008 at the Lausanne hospital. The genetic comparison of these isolates revealed that their genomes are extremely closely related. Yet, a few more important genetic changes, such as the replacement of a plasmid, the loss of large fragments of DNA, or the insertion of transposases, were observed. These transfers of mobile genetic elements shaped the evolution of the ST228 lineage that spread within the Lausanne hospital. Nevertheless, although the strains analyzed differed in their dynamics, we have not been able to link a particular genetic element with spreading success. Finally, the present study showed that new sequencing technologies improve considerably the quality and quantity of information obtained for a single strain; but this information is still difficult to interpret and important investments are required for the technology to become accessible for routine investigations.

Molecular and isotopic characterization of biomarkers in the Frick Swiss Jura sediments: A palaeoenvironmental reconstruction on the northern Tethys margin

Molecular and stable carbon isotope compositions of source-specific hydrocarbons have been used to reconstruct palaeoenvironmental conditions during deposition of the Middle Hettangian to Upper Sinemurian sediments on the northern epicontinental Tethys margin, Frick Swiss Jura. Increasing algal, cyanobacterial and phytoplanktonic (i.e., dinoflagellate) contributions associated with the C-13-enrichment of cyanobacteria derivatives (i.e., hopanes and monomethylalkanes) suggest enhanced primary productivity upsection. This is related to the C-13-enrichment of dissolved CO2 in the upper layers and the progressive increase of depth and oxygenation of the water column. In the Middle Hettangian shallow-water environments (lagoon), the occurrence of green sulfur bacteria (Chlorobiaceae) derivatives indicates that the lower part of the water column was strictly anoxic and rich in H2S. Since these bacteria require very low light intensity to grow, these euxinic conditions may be extended up to the photic zone, allowing for anaerobic photosynthesis. Light penetration depth is most likely reduced by high productivity and/or turbidity in the photic zone. In these sediments, C-13-depleted hopanoids (-39.5 parts per thousand) are most likely associated with phototrophic purple sulfur bacteria utilizing isotopically light organic carbon at the base of the aerobic zone. These purple sulfur bacteria may have consumed the H2S used by Chlorobiaceae in the deeper layers and thus, sustained the algae and cyanobacteria productivity in the upper layers. The C-13-depleted carbonate (-13.3 parts per thousand) may be partially related to the anaerobic oxidation of the organic matter during bacterial sulfate-reduction. (c) 2006 Elsevier Ltd. All rights reserved.

SwissSidechain: a molecular and structural database of non-natural sidechains.

Amino acids form the building blocks of all proteins. Naturally occurring amino acids are restricted to a few tens of sidechains, even when considering post-translational modifications and rare amino acids such as selenocysteine and pyrrolysine. However, the potential chemical diversity of amino acid sidechains is nearly infinite. Exploiting this diversity by using non-natural sidechains to expand the building blocks of proteins and peptides has recently found widespread applications in biochemistry, protein engineering and drug design. Despite these applications, there is currently no unified online bioinformatics resource for non-natural sidechains. With the SwissSidechain database (http://www.swisssidechain.ch), we offer a central and curated platform about non-natural sidechains for researchers in biochemistry, medicinal chemistry, protein engineering and molecular modeling. SwissSidechain provides biophysical, structural and molecular data for hundreds of commercially available non-natural amino acid sidechains, both in l- and d-configurations. The database can be easily browsed by sidechain names, families or physico-chemical properties. We also provide plugins to seamlessly insert non-natural sidechains into peptides and proteins using molecular visualization software, as well as topologies and parameters compatible with molecular mechanics software.

Molecular genetics of "PRPF31" dominant mutations linked to retinitis pigmentosa

ABSTRACT : The retina is one of the most important human sensory tissues since it detects and transmits all visual information from the outside world to the brain. Retinitis pigmentosa (RP) is the name given to a group of inherited diseases that affect specifically the photoreceptors present in the retina and in many instances lead to blindness. Dominant mutations in PRPF31, a gene that encodes for a pre-mRNA splicing factor, cause retinitis pigmentosa with reduced penetrance. We functionally investigated a novel mutation, identified in a large family with autosomal dominant RP, and 7 other mutations, substitutions and microdeletions, in 12 patients from 7 families with PRPF31-linked RP. Seven mutations lead to PRPF31 mRNA with premature stop codons and one to mRNA lacking the exon containing the initiation codon. Quantification of PRPF31 mRNA and protein levels revealed a significant reduction in cell lines derived from patients, compared to non carriers of mutations in PRPF31. Allelic quantification of PRPF31 mRNA indicated that the level of mutated mRNA is very low compared to wild-type mRNA. No mutant protein was detected and the subnuclear localization of wild-type PRPF31 remains the same in cell lines from patients and controls. Blocking nonsense-mediated mRNA decay in cell lines derived from patients partially restored PRPF31 mutated mRNA but derived proteins were still undetectable, even when protein degradation pathways were inhibited. Our results demonstrated that the vast majority of PRPF31 mutations result in null alleles, since they are subject to surveillance mechanisms that degrade mutated mRNA and possibly block its translation. Altogether, these data indicate that the likely cause of PRPF31-linked RP is haploinsufficiency, rather than a dominant negative effect. Penetrance of PRPF31 mutations has been previously demonstrated to be inversely correlated with the level of PRPF31 mRNA, since high expression of wild-type PRPF31 mRNA protects from the disease. Consequently, we have investigated the genetic modifiers that control the expression of PRPF31 by quantifying PRPF31 mRNA levels in cell lines derived from 200 individuals from 15 families representative of the general population. By linkage analyses we identified a 8.2Mb-region on chromosome 14q21-23 that contains a gene involved in the modulation of PRPF31 expression. We also assessed apreviously-mapped penetrance factor invariably located on the wild-type allele and linked to the PRPF31 locus in asymptomatic patients from different families with RP. We demonstrated that this modifier increases the expression of both PRPF31 alleles already at the pre-mRNA level. Finally, our data suggest that PRPF31 mRNA expression and consequently the penetrance of PRPF31 mutations is modulated by at least 2 diffusible compounds, which act on both PRPF31 alleles during their transcription.

Homer1a is a core brain molecular correlate of sleep loss.

Sleep is regulated by a homeostatic process that determines its need and by a circadian process that determines its timing. By using sleep deprivation and transcriptome profiling in inbred mouse strains, we show that genetic background affects susceptibility to sleep loss at the transcriptional level in a tissue-dependent manner. In the brain, Homer1a expression best reflects the response to sleep loss. Time-course gene expression analysis suggests that 2,032 brain transcripts are under circadian control. However, only 391 remain rhythmic when mice are sleep-deprived at four time points around the clock, suggesting that most diurnal changes in gene transcription are, in fact, sleep-wake-dependent. By generating a transgenic mouse line, we show that in Homer1-expressing cells specifically, apart from Homer1a, three other activity-induced genes (Ptgs2, Jph3, and Nptx2) are overexpressed after sleep loss. All four genes play a role in recovery from glutamate-induced neuronal hyperactivity. The consistent activation of Homer1a suggests a role for sleep in intracellular calcium homeostasis for protecting and recovering from the neuronal activation imposed by wakefulness.

Phosphatidylinositol 4,5-bisphosphate clusters act as molecular beacons for vesicle recruitment.

Synaptic-vesicle exocytosis is mediated by the vesicular Ca(2+) sensor synaptotagmin-1. Synaptotagmin-1 interacts with the SNARE protein syntaxin-1A and acidic phospholipids such as phosphatidylinositol 4,5-bisphosphate (PIP2). However, it is unclear how these interactions contribute to triggering membrane fusion. Using PC12 cells from Rattus norvegicus and artificial supported bilayers, we show that synaptotagmin-1 interacts with the polybasic linker region of syntaxin-1A independent of Ca(2+) through PIP2. This interaction allows both Ca(2+)-binding sites of synaptotagmin-1 to bind to phosphatidylserine in the vesicle membrane upon Ca(2+) triggering. We determined the crystal structure of the C2B domain of synaptotagmin-1 bound to phosphoserine, allowing development of a high-resolution model of synaptotagmin bridging two different membranes. Our results suggest that PIP2 clusters organized by syntaxin-1 act as molecular beacons for vesicle docking, with the subsequent Ca(2+) influx bringing the vesicle membrane close enough for membrane fusion.

Gene expression mapping in neuropathic pain : molecular plasticity in nociceptors and superficial dorsal horn

RESUME : La douleur neuropathique est le résultat d'une lésion ou d'un dysfonctionnement du système nerveux. Les symptômes qui suivent la douleur neuropathique sont sévères et leur traitement inefficace. Une meilleure approche thérapeutique peut être proposée en se basant sur les mécanismes pathologiques de la douleur neuropathique. Lors d'une lésion périphérique une douleur neuropathique peut se développer et affecter le territoire des nerfs lésés mais aussi les territoires adjacents des nerfs non-lésés. Une hyperexcitabilité des neurones apparaît au niveau des ganglions spinaux (DRG) et de la corne dorsale (DH) de la moelle épinière. Le but de ce travail consiste à mettre en évidence les modifications moléculaires associées aux nocicepteurs lésés et non-lésés au niveau des DRG et des laminae I et II de la corne dorsale, là où l'information nociceptive est intégrée. Pour étudier les changements moléculaires liés à la douleur neuropathique nous utilisons le modèle animal d'épargne du nerf sural (spared nerve injury model, SNI) une semaine après la lésion. Pour la sélection du tissu d'intérêt nous avons employé la technique de la microdissection au laser, afin de sélectionner une sous-population spécifique de cellules (notamment les nocicepteurs lésés ou non-lésés) mais également de prélever le tissu correspondant dans les laminae superficielles. Ce travail est couplé à l'analyse à large spectre du transcriptome par puce ADN (microarray). Par ailleurs, nous avons étudié les courants électriques et les propriétés biophysiques des canaux sodiques (Na,,ls) dans les neurones lésés et non-lésés des DRG. Aussi bien dans le système nerveux périphérique, entre les neurones lésés et non-lésés, qu'au niveau central avec les aires recevant les projections des nocicepteurs lésés ou non-lésés, l'analyse du transcriptome montre des différences de profil d'expression. En effet, nous avons constaté des changements transcriptionnels importants dans les nocicepteurs lésés (1561 gènes, > 1.5x et pairwise comparaison > 77%) ainsi que dans les laminae correspondantes (618 gènes), alors que ces modifications transcriptionelles sont mineures au niveau des nocicepteurs non-lésés (60 gènes), mais important dans leurs laminae de projection (459 gènes). Au niveau des nocicepteurs, en utilisant la classification par groupes fonctionnels (Gene Ontology), nous avons observé que plusieurs processus biologiques sont modifiés. Ainsi des fonctions telles que la traduction des signaux cellulaires, l'organisation du cytosquelette ainsi que les mécanismes de réponse au stress sont affectés. Par contre dans les neurones non-lésés seuls les processus biologiques liés au métabolisme et au développement sont modifiés. Au niveau de la corne dorsale de la moelle, nous avons observé des modifications importantes des processus immuno-inflammatoires dans l'aire affectée par les nerfs lésés et des changements associés à l'organisation et la transmission synaptique au niveau de l'aire des nerfs non-lésés. L'analyse approfondie des canaux sodiques a démontré plusieurs changements d'expression, principalement dans les neurones lésés. Les analyses fonctionnelles n'indiquent aucune différence entre les densités de courant tétrodotoxine-sensible (TTX-S) dans les neurones lésés et non-lésés même si les niveaux d'expression des ARNm des sous-unités TTX-S sont modifiés dans les neurones lésés. L'inactivation basale dépendante du voltage des canaux tétrodotoxine-insensible (TTX-R) est déplacée vers des potentiels positifs dans les cellules lésées et non-lésées. En revanche la vitesse de récupération des courants TTX-S et TTX-R après inactivation est accélérée dans les neurones lésés. Ces changements pourraient être à l'origine de l'altération de l'activité électrique des neurones sensoriels dans le contexte des douleurs neuropathiques. En résumé, ces résultats suggèrent l'existence de mécanismes différenciés affectant les neurones lésés et les neurones adjacents non-lésés lors de la mise en place la douleur neuropathique. De plus, les changements centraux au niveau de la moelle épinière qui surviennent après lésion sont probablement intégrés différemment selon la perception de signaux des neurones périphériques lésés ou non-lésés. En conclusion, ces modulations complexes et distinctes sont probablement des acteurs essentiels impliqués dans la genèse et la persistance des douleurs neuropathiques. ABSTRACT : Neuropathic pain (NP) results from damage or dysfunction of the peripheral or central nervous system. Symptoms associated with NP are severe and difficult to treat. Targeting NP mechanisms and their translation into symptoms may offer a better therapeutic approach.Hyperexcitability of the peripheral and central nervous system occurs in the dorsal root ganglia (DRG) and the dorsal horn (DH) of the spinal cord. We aimed to identify transcriptional variations in injured and in adjacent non-injured nociceptors as well as in corresponding laminae I and II of DH receiving their inputs.We investigated changes one week after the injury induced by the spared nerve injury model of NP. We employed the laser capture microdissection (LCM) for the procurement of specific cell-types (enrichment in nociceptors of injured/non-injured neurons) and laminae in combination with transcriptional analysis by microarray. In addition, we studied functionál properties and currents of sodium channels (Nav1s) in injured and neighboring non-injured DRG neurons.Microarray analysis at the periphery between injured and non-injured DRG neurons and centrally between the area of central projections from injured and non-injured neurons show significant and differential expression patterns. We reported changes in injured nociceptors (1561 genes, > 1.5 fold, >77% pairwise comparison) and in corresponding DH laminae (618 genes), while less modifications occurred in non-injured nociceptors (60 genes) and in corresponding DH laminae (459 genes). At the periphery, we observed by Gene Ontology the involvement of multiple biological processes in injured neurons such as signal transduction, cytoskeleton organization or stress responses. On contrast, functional overrepresentations in non-injured neurons were noted only in metabolic or developmentally related mechanisms. At the level of superficial laminae of the dorsal horn, we reported changes of immune and inflammatory processes in injured-related DH and changes associated with synaptic organization and transmission in DH corresponding to non-injured neurons. Further transcriptional analysis of Nav1s indicated several changes in injured neurons. Functional analyses of Nav1s have established no difference in tetrodotoxin-sensitive (TTX-S) current densities in both injured and non-injured neurons, despite changes in TTX-S Nav1s subunit mRNA levels. The tetrodotoxin-resistant (TTX-R) voltage dependence of steady state inactivation was shifted to more positive potentials in both injured and non-injured neurons, and the rate of recovery from inactivation of TTX-S and TTX-R currents was accelerated in injured neurons. These changes may lead to alterations in neuronal electrogenesis. Taken together, these findings suggest different mechanisms occurring in the injured neurons and the adjacent non-injured ones. Moreover, central changes after injury are probably driven in a different manner if they receive inputs from injured or non-injured neurons. Together, these distinct and complex modulations may contribute to NP.

Molecular definition of inflammasomes

Inflammasomes are crucial actors of the innate immunity. They consist of cytoplasmic multiprotein complexes controlling the biological activities of the inflammatory cytokines IL-1ß and IL-18. Inflammasome assembly depends on protein domain interaction. In this chapter, we focus on the biochemical aspects characterizing the components of the inflammasomes and their assembly into a high moelcuar weight protelytic complex.

Traitement de la coxarthrose par des prothèses totales de hanche sur mesure [Treatment of coxarthrosis with fitted total hip prostheses replacement]

The treatment of hip osteoarthritis with total hip arthroplasty has continuously evolved since it was first introduced in the sixties. The problem of aseptic loosening of the cemented prostheses, mainly in young active patients, has stimulated two different types of research: on one side the improvement of cementing techniques and on the other side the development of cementless osteoinegrable implants. We discuss the problems of these cementless hip prostheses. Recently published anatomic and biomechanic studies have led to the development of personalized custom femoral stems for each patient. The conception technique and first clinical results are described.

Multi-segment foot kinematics after total ankle replacement and ankle arthrodesis during relatively long-distance gait.

This study aimed to investigate the influence of ankle osteoarthritis (AOA) treatments, i.e., ankle arthrodesis (AA) and total ankle replacement (TAR), on the kinematics of multi-segment foot and ankle complex during relatively long-distance gait. Forty-five subjects in four groups (AOA, AA, TAR, and control) were equipped with a wearable system consisting of inertial sensors installed on the tibia, calcaneus, and medial metatarsals. The subjects walked 50-m twice while the system measured the kinematic parameters of their multi-segment foot: the range of motion of joints between tibia, calcaneus, and medial metatarsals in three anatomical planes, and the peaks of angular velocity of these segments in the sagittal plane. These parameters were then compared among the four groups. It was observed that the range of motion and peak of angular velocities generally improved after TAR and were similar to the control subjects. However, unlike AOA and TAR, AA imposed impairments in the range of motion in the coronal plane for both the tibia-calcaneus and tibia-metatarsals joints. In general, the kinematic parameters showed significant correlation with established clinical scales (FFI and AOFAS), which shows their convergent validity. Based on the kinematic parameters of multi-segment foot during 50-m gait, this study showed significant improvements in foot mobility after TAR, but several significant impairments remained after AA.

Narcolepsy and familial advanced sleep-phase syndrome: molecular genetics of sleep disorders.

Sleep disorders are very prevalent and represent an emerging worldwide epidemic. However, research into the molecular genetics of sleep disorders remains surprisingly one of the least active fields. Nevertheless, rapid progress is being made in several prototypical disorders, leading recently to the identification of the molecular pathways underlying narcolepsy and familial advanced sleep-phase syndrome. Since the first reports of spontaneous and induced loss-of-function mutations leading to hypocretin deficiency in human and animal models of narcolepsy, the role of this novel neurotransmission pathway in sleep and several other behaviors has gained extensive interest. Also, very recent studies using an animal model of familial advanced sleep-phase syndrome shed new light on the regulation of circadian rhythms.

Molecular neuro-oncology in clinical practice: a new horizon.

Primary brain tumours are heterogeneous in histology, genetics, and outcome. Although WHO's classification of tumours of the CNS has greatly helped to standardise diagnostic criteria worldwide, it does not consider the substantial progress that has been made in the molecular classification of many brain tumours. Recent practice-changing clinical trials have defined a role for routine assessment of MGMT promoter methylation in glioblastomas in elderly people, and 1p and 19q codeletions in anaplastic oligodendroglial tumours. Moreover, large-scale molecular profiling approaches have identified new mutations in gliomas, affecting IDH1, IDH2, H3F3, ATRX, and CIC, which has allowed subclassification of gliomas into distinct molecular subgroups with characteristic features of age, localisation, and outcome. However, these molecular approaches cannot yet predict patients' benefit from therapeutic interventions. Similarly, transcriptome-based classification of medulloblastoma has delineated four variants that might now be candidate diseases in which to explore novel targeted agents.

Molecular pathways involved in the antineoplastic effects of calcitriol on insulinoma cells.

We have previously reported that in tumorigenic pancreatic beta-cells, calcitriol exerts a potent antitumorigenic effect by inducing apoptosis, cell growth inhibition, and reduction of solid beta-cell tumors. Here we have studied the molecular pathways involved in the antineoplastic activity of calcitriol on mouse insulinoma beta TC(3) cells, mouse insulinoma beta TC expressing or not expressing the oncogene p53, and beta TC-tet cells overexpressing or not the antiapoptotic gene Bcl2. Our results indicate that calcitriol-induced apoptosis was dependent on the function of p53 and was associated with a biphasic increase in protein levels of transcription factor nuclear factor-kappa B. Calcitriol decreased cell viability by about 40% in p53-retaining beta TC and in beta TC(3) cells; in contrast, beta TC p53(-/-) cells were only minimally affected. Calcitriol-induced cell death was regulated by members of the Bcl-2 family of apoptosis regulatory proteins, as shown by calcitriol-induced up-regulation of proapoptotic Bax and Bak and the lack of calcitriol-induced cytotoxicity in Bcl-2-overexpressing insulinoma cells. Moreover, calcitriol-mediated arrest of beta TC(3) cells in the G(1) phase of the cell cycle was associated with the abnormal expression of p21 and G(2)/M-specific cyclin B2 genes and involved the DNA damage-inducible factor GADD45. Finally, in beta TC(3) cells, calcitriol modulated the expression of IGF-I and IGF-II genes. In conclusion, these findings contribute to the understanding of the antitumorigenic effects of calcitriol on tumorigenic pancreatic beta-cells and further support the rationale of its utilization in the treatment of patients with malignant insulinomas.