[Validation of the questionnaire for assessing the childbirth experience (QACE) in a French population]. / Validation de l'échelle d'évaluation du vécu de l'accouchement (QEVA) auprès d'une population française.

BACKGROUND: A woman's negative perception of her subjective childbirth experience can have consequences on the mother's psychological state and on early mother-baby relationships. To date, there is no validated tool in France allowing to evaluate childbirth experience in a multidimensional way. The aim of this study is to validate the Questionnaire Assessing the Childbirth Experience (QEVA) in a French sample of mothers. This tool was developed in a previous study where the authors combined 25 items into 6 dimensions: representations and expectations, sensory perceptions, feeling of control, perceived social support (medical staff and partner), emotions (positive and negative) and first moments with the baby. METHODS: The sample included 256 women recruited in a maternity ward. Sociodemographic and obstetric characteristics of our sample were compared to those of the French national perinatal survey. The structure of the QEVA with 17 items was explored by an exploratory structural equation modeling (ESEM). An analysis of the internal consistency was conducted on the sub-scores of the identified factors, and the concurrent validity was assessed with the Peri-traumatic Distress Inventory (PDI) through a correlation and its associated t-test. RESULTS: The characteristics of our sample and those of the national perinatal survey do not differ on age, marital status, parity, cannabis use, infertility treatment, epidural and baby weight, in favour of the good representativeness of our sample. The study of the QEVA structure revealed a 4-dimensional structure. Analysis of the psychometric qualities showed a good internal consistency, with an observed alpha value ranging from 0.69 to 0.86. The QEVA also shows a good concurrent validity with the peri-traumatic distress scores (r=0.51). CONCLUSION: To date, the QEVA is the first standardized tool allowing a multidimensional evaluation of the subjective experience of childbirth. It has been validated on a French population using an exploratory structural equation modeling. This tool, which is simple to use and well accepted by mothers, enables health professionals not only to screen mothers experiencing difficult childbirth and in need of support, but also to adapt health care according to the dimensions of the birth experience and its associated difficulties (emotions during the birth, interactions with health professionals, first moments with the baby, or post-partum emotions).

Signaling switch of the urotensin II vasosactive peptide GPCR: prototypic chemotaxic mechanism in glioma.

Multiform glioblastomas (GBM) are the most frequent and aggressive primary brain tumors in adults. The poor prognosis is due to neo-angiogenesis and cellular invasion, processes that require complex chemotaxic mechanisms involving motility, migration and adhesion. Understanding these different cellular events implies identifying receptors and transduction pathways that lead to and promote either migration or adhesion. Here we establish that glioma express the vasoactive peptide urotensin II (UII) and its receptor UT and that UT-mediated signaling cascades are involved in glioma cell migration and adhesion. Components of the urotensinergic systems, UII and UT, are widely expressed in patient-derived GBM tissue sections, glioma cell lines and fresh biopsy explants. Interestingly, gradient concentrations of UII produced chemoattracting migratory/motility effects in glioma as well as HEK293 cells expressing human UT. These effects mainly involved the G13/Rho/rho kinase pathway while partially requiring Gi/o/PI3K components. In contrast, we observed that homogeneous concentrations of UII drastically blocked cell motility and stimulated cell-matrix adhesions through a UT/Gi/o signaling cascade, partially involving phosphatidylinositol-3 kinase. Finally, we provide evidence that, in glioma cells, homogeneous concentration of UII allowed translocation of Gα13 to the UT receptor at the plasma membrane and increased actin stress fibers, lamellipodia formation and vinculin-stained focal adhesions. UII also provoked a re-localization of UT precoupled to Gαi in filipodia and initiated integrin-stained focal points. Altogether, these findings suggest that UT behaves as a chemotaxic receptor, relaying a signaling switch between directional migration and cell adhesion under gradient or homogeneous concentrations, thereby redefining sequential mechanisms affecting tumor cells during glioma invasion. Taken together, our results allow us to propose a model in order to improve the design of compounds that demonstrate signaling bias for therapies that target specifically the Gi/o signaling pathway.

Evolution of class A G-protein-coupled receptors: implications for molecular modeling.

Class A or rhodopsin-like G-protein-coupled receptors (GPCRs) constitute the largest transmembrane receptor family of the human genome. Because of their biological and pharmaceutical importance, the evolutionary history of these receptors has been widely studied. Most studies agree on the classification of the 700 members of this family into a dozen of sub-families. However, the relationship between these sub-families remains controversial and the molecular processes that drove the evolution and diversification of such a large family have still to be determined. We review here the evolutionary analyses carried out on class A GPCRs either by phylogenetic methods or by multidimensional scaling (MDS). We detail the key molecular events driving the evolution of this receptor family. We analyze these events in view of the recently resolved crystal structures of GPCRs and we discuss the usefulness of evolutionary information to help molecular modeling.

The ciliary neurotrophic factor receptor alpha component induces the secretion of and is required for functional responses to cardiotrophin-like cytokine.

Ciliary neurotrophic factor (CNTF) is involved in the survival of a number of different neural cell types, including motor neurons. CNTF functional responses are mediated through a tripartite membrane receptor composed of two signalling receptor chains, gp130 and the leukaemia inhibitory factor receptor (LIFR), associated with a non-signalling CNTF binding receptor alpha component (CNTFR). CNTFR-deficient mice show profound neuronal deficits at birth, leading to a lethal phenotype. In contrast, inactivation of the CNTF gene leads only to a slight muscle weakness, mainly during adulthood, suggesting that CNTFR binds to a second ligand that is important for development. Modelling studies of the interleukin-6 family member cardiotrophin-like cytokine (CLC) revealed structural similarities with CNTF, including the conservation of a site I domain involved in binding to CNTFR. Co-expression of CLC and CNTFR in mammalian cells generates a secreted composite cytokine, displaying activities on cells expressing the gp130-LIFR complex on their surface. Correspondingly, CLC-CNTFR activates gp130, LIFR and STAT3 signalling components, and enhances motor neuron survival. Together, these observations demonstrate that CNTFR induces the secretion of CLC, as well as mediating the functional responses of CLC.

Backbone dynamics of Tet repressor alpha8intersectionalpha9 loop.

A set of single Trp mutants of class B Tet repressor (TetR), in which Trp residues are located from positions 159 to 167, has been engineered to investigate the dynamics of the loop joining the alpha-helices 8 and 9. The fluorescence anisotropy decay of most mutants can be described by the sum of three exponential components. The longest rotational correlation time, 30 ns at 10 degrees C, corresponds to the overall rotation of the protein. The shortest two components, on the subnanosecond and nanosecond time scale, are related to internal motions of the protein. The initial anisotropy, in the 0.16-0.22 range, indicates the existence of an additional ultrafast motion on the picosecond time scale. Examination of physical models for underlying motions indicates that librational motions of the Trp side chain within the rotameric chi(1) x chi(2) potential wells contribute to the picosecond depolarization process, whereas the subnanosecond and nanosecond depolarization processes are related to backbone dynamics. In the absence of inducer, the order parameters of these motions, about 0.90 and 0.80 for most positions, indicate limited flexibility of the loop backbone. Anhydrotetracycline binding to TetR induces an increased mobility of the loop on the nanosecond time scale. This suggests that entropic factors might play a role in the mechanism of allosteric transition.

Identification of a gp130 cytokine receptor critical site involved in oncostatin M response.

Gp130 cytokine receptor is involved in the formation of multimeric functional receptors for interleukin-6 (IL-6), IL-11, leukemia inhibitory factor (LIF), oncostatin M (OSM), ciliary neurotrophic factor, and cardiotrophin-1. Cloning of the epitope recognized by an OSM-neutralizing anti-gp130 monoclonal antibody identified a portion of gp130 receptor localized in the EF loop of the cytokine binding domain. Site-directed mutagenesis of the corresponding region was carried out by alanine substitution of residues 186-198. To generate type 1 or type 2 OSM receptors, gp130 mutants were expressed together with either LIF receptor beta or OSM receptor beta. When positions Val-189/Tyr-190 and Phe-191/Val-192 were alanine-substituted, Scatchard analyses indicated a complete abrogation of OSM binding to both type receptors. Interestingly, binding of LIF to type 1 receptor was not affected, corroborating the notion that in this case gp130 mostly behaves as a converter protein rather than a binding receptor. The present study demonstrates that positions 189-192 of gp130 cytokine binding domain are essential for OSM binding to both gp130/LIF receptor beta and gp130/OSM receptor beta heterocomplexes.

Trp scanning analysis of Tet repressor reveals conformational changes associated with operator and anhydrotetracycline binding.

We analysed the conformational states of free, tet operator-bound and anhydrotetracycline-bound Tet repressor employing a Trp-scanning approach. The two wild-type Trp residues in Tet repressor were replaced by Tyr or Phe and single Trp residues were introduced at each of the positions 162-173, representing part of an unstructured loop and the N-terminal six residues of alpha-helix 9. All mutants retained in vivo inducibility, but anhydrotetracycline-binding constants were decreased up to 7.5-fold when Trp was in positions 169, 170 and 173. Helical positions (168-173) differed from those in the loop (162-167) in terms of their fluorescence emission maxima, quenching rate constants with acrylamide and anisotropies in the free and tet operator-complexed proteins. Trp fluorescence emission decreased drastically upon atc binding, mainly due to energy transfer. For all proteins, either free, tet operator bound or anhydrtetracycline-bound, mean fluorescence lifetimes were determined to derive quenching rate constants. Solvent-accessible surfaces of the respective Trp side chains were calculated and compared with the quenching rate constants in the anhydrotetracycline-bound complexes. The results support a model, in which residues in the loop become more exposed, whereas residues in alpha-helix 9 become more buried upon the induction of TetR by anhydrotetracycline.

Role of Trp-187 in the annexin V-membrane interaction: a molecular mechanics analysis.

The minimized energy mapping of annexin V Trp-187 chi1 x chi2 isomerization supports the existence of two preferential rotameric orientations of the Trp side chain upon annexin V binding to membranes, in agreement with the time-resolved fluorescence results. They correspond to the perpendicular trans (-173 degrees, 73 degrees) and g- (-71 degrees, 83 degrees) rotamers and represent 59 and 28% of the population, respectively. The analysis of their local environment makes it possible to assign the trans rotamer to the long component and the g- rotamer to the short component of the biexponential fluorescence decay. The orientation of these rotamers relative to the protein core suggests a dual role for Trp-187, which might be involved both in the interaction with the phospholipid bilayer and in the formation of the annexin V 2-D array at the surface of the membrane.

Structural investigation of Tet repressor loop 154-167: a time-resolved fluorescence study of three single Trp mutants.

We have studied the time-resolved fluorescence of three engineered Tet repressor (TetR) mutants bearing a single Trp residue at positions 162, 163, and 165 in the C-terminal part of the loop joining helices 8 and 9. Detailed analysis indicates that, at 20 degrees, the fluorescence decay of each Trp can be described as the sum of three exponential components with lifetimes in the 1-, 3-, and 6-ns range. Emission wavelength and temperature dependence studies are consistent with a model in which these components are due to the existence of three classes of Trp residues non-interconverting on the nanosecond timescale. Within the framework of the rotamer model, the weak temperature dependence of the lifetimes strongly suggests that the secondary structure of the loop, at least in the 162-165 range, is not altered with temperature. The equilibrium between the rotamers is characterized by an enthalpy-entropy compensation effect which strongly suggests the involvement of background structural regions of TetR in the thermodynamics of the process. The very high deltaH degrees and TdeltaS degrees observed (up to 18 kcal/ mol) should reflect the temperature-dependent conformational change of a large part of the protein which would alter the rotamer distribution of the Trp residues. Taken together, our results are consistent with the existence of (at least) two conformations of the loop and suggest a model for loop motion.

Molecular mechanics analysis of Tet repressor TRP-43 fluorescence.

A 35% decrease in the fluorescence intensity of F75 TetR Trp-43 was observed upon binding of the tetracycline derivative 5a,6-anhydrotetracycline (AnTc) to the repressor. The fluorescence decay of Trp-43 in F75 TetR and in its complex with AnTc could be described by the sum of three exponential components, with lifetimes of about 6, 3, and 0.3 ns. The amplitudes, however, were markedly altered upon binding. The minimized energy mapping of Trp-43 chi 1 x chi 2 isomerization clearly indicated the existence of three main potential wells at positions (-160 degrees, -90 degrees) (rotamer I), (-170 degrees, 90 degrees) (rotamer II), and (-70, 150 degrees) (rotamer III). Our study of Trp-43 environment for each of the three rotamers suggests that the longest decay component may be assigned to rotamer II, the middle-lived component to rotamer I, and the subnanosecond component to rotamer III. The origin of the changes in the rotamer distribution upon AnTc binding is discussed. Anisotropy decays are also discussed within the framework of the rotamer model.

Spectroscopic investigation of Tet repressor tryptophan-43 upon specific and nonspecific DNA binding.

The F75 Tet repressor mutant (F75 TetR) contains a single tryptophan residue located at position 43 in the operator recognition alpha-helix. Previous studies [Hansen, D., & Hillen, W. (1987) J. Biol. Chem. 262, 12269-12274] have shown that the fluorescence intensity of this residue is dramatically reduced upon operator binding. In order to determine the origin of this quenching and the role of Trp-43 in the binding mechanism, we have investigated its fluorescence properties upon F75 TetR binding to a tet operator containing 76 bp DNA fragment (specific binding) and to sheared calf thymus DNA (nonspecific binding). Trp-43 steady-state fluorescence intensity was quenched by 72% upon specific binding and by 45% upon nonspecific binding. These fluorescence intensity decreases were not accounted for by similar decreases in the respective fluorescence lifetimes. The apparent quenching calculated from the average lifetimes was about 0.33 in both binding modes. This shows the presence of a static quenching process, clearly favored upon specific binding as compared to nonspecific binding. This is consistent with stacking interactions between Trp-43 and the DNA bases, as suggested by molecular graphics [Baumeister, R., Helbl, V., & Hillen, W. (1992) J. Mol. Biol. 226, 1257-1270]. The equilibrium constant between nonfluorescent and fluorescent tryptophan residues was 5 times higher upon binding to specific DNA than to nonspecific DNA. The preferential static quenching of Trp-43 in the specific complex suggests that stacking interactions might contribute to the specific binding mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

Fluorescence of a tryptophan bearing peptide from smooth muscle myosin light chain kinase upon binding to two closely related calmodulins.

We have investigated the fluorescence of a calmodulin binding peptide (AS19) based on the sequence of the calmodulin binding domain of smooth muscle myosin light chain kinase and bearing a tryptophan residue at position 5 upon binding to two closely related calmodulins. The emission maximum of peptide AS19 bound to the engineered SYNCAM calmodulin was 318 nm and a vibrational structure was clearly apparent. The emission maximum of peptide AS19 bound to chicken gizzard calmodulin (ChG CaM) was 327 nm and its spectrum was featureless. Red edge excitation effect supports the assumption that the polarity of Trp-5 environment is larger in the complex with ChG CaM than with SYNCAM, in agreement with fluorescence spectra. Time-resolved fluorescence and anisotropy measurements showed that, in both complexes, the tryptophan emitting state was 1La. The X-ray structure of the calmodulin-peptide complex has been resolved (W. E. Meador, A. R. Means, and F. A. Quiocho, 1992, Science 257, 1251-1255). The Trp binding site has been characterized. It differs by a single-point mutation between the two calmodulins: Met-144 of ChG CaM has been replaced by Val in SYNCAM. This suggests that the spectral relaxation of Trp-5 in the complex with ChG CaM as compared to SYNCAM is due to the polarizability of the sulfur atom containing Met side chain that is higher than that of Val. This provides an ideal system to investigate the origin of the Stokes shift of the indole moiety in proteins.

Structural analysis of the operator binding domain of Tn10-encoded Tet repressor: a time-resolved fluorescence and anisotropy study.

An engineered Tn10-encoded Tet repressor, bearing a single Trp residue at position 43, in the putative alpha-helix-turn-alpha-helix motif of the operator binding domain, was studied by time-resolved fluorescence and anisotropy. Fluorescence intensity decay data suggested the existence of two classes of Trp-43, defined by different lifetimes. Analysis of anisotropy data were consistent with a model in which each class was defined by a different lifetime, rotational correlation time, and fluorescence emission maximum. The long-lifetime class had a red-shifted spectrum, similar to that of tryptophan zwitterion in water, and a short rotational correlation time. In contrast, the spectrum of the short-lifetime class was blue-shifted 10 nm compared to that of the long-lifetime class. Its correlation time was similar to that of the protein, which showed that Trp in this class was entirely constrained. Trp in this latter class could not be quenched by iodide, whereas most of the long-lifetime class was easily accessible. Presence of disruptive agents, such as 1 M GuCl or 3 M KCl, did not alter markedly the lifetimes but increased the weight of the short-lifetime component. In the same time, the rotational correlation time of this component was dramatically reduced. Taken together, our data suggest that the long-lifetime class could correspond to the tryptophan residues exposed to solvent whereas the short-lifetime class would correspond to the tryptophan residues embedded inside the hydrophobic core holding the helix-turn-helix motif. Destabilization of hydrophobic interactions would lead to an increase in the weight of the latter class for entropic reasons. Analysis of the fluorescence parameters of Trp-43 could provide structural information on the operator binding domain of Tet repressor.

Fluorescence analysis of calmodulin mutants containing tryptophan: conformational changes induced by calmodulin-binding peptides from myosin light chain kinase and protein kinase II.

Peptide-induced conformational changes in five isofunctional mutants of calmodulin (CaM), each bearing a single tryptophan residue either at the seventh position of each of the four calcium-binding loops (i.e., amino acids 26, 62, 99, and 135) or in the central helix (amino acid 81) were studied by using fluorescence spectroscopy. The peptides RS20F and RS20CK correspond to CaM-binding amino acid sequence segments of either nonmuscle myosin light chain kinase (nmMLCK) or calmodulin-dependent protein kinase II (CaMPK-II), respectively. Both steady-state and time-resolved fluorescence data were collected from the various peptide-CaM complexes. Steady-state fluorescence intensity measurements indicated that, in the presence of an excess of calcium, both peptides bind to the calmodulin mutants with a 1:1 stoichiometry. The tryptophans located in loops I and IV exhibited red-shifted emission maxima (356 nm), high quantum yields (0.3), and long average lifetimes (6 ns). They responded in a similar manner to peptide binding, by only slight changes in their fluorescence features. In contrast, the fluorescence intensity of the tryptophans in loops II and III decreased markedly, and their fluorescence spectrum was blue-shifted upon peptide binding. Analysis of the tryptophan fluorescence decay of the last mentioned calmodulins supports a model in which the equilibrium between two (Trp-99) or three (Trp-62) states of these tryptophan residues, each characterized by a different lifetime, was altered toward the blue-shifted short lifetime component upon peptide binding. Taken together, these data provide new evidence that both lobes of calmodulin are involved in peptide binding. Both peptides induced similar changes in the fluorescence properties of the tryptophan residues located in the calcium-binding loops, with the exception of calmodulin with Trp-135. For this last mentioned calmodulin, slight differences were observed. Tryptophan in the central helix responded differently to RS20F and RS20CK binding. RS20F binding induced a red-shift in the emission maximum of Trp-81 while RS20CK induced a blue-shift. The quenching rate of Trp-81 by iodide was slightly reduced upon RS20CK binding, while RS20F induced a 2-fold increase. These results provide evidence that the environment of Trp-81 is different in each case and are, therefore, consistent with the hypothesis that the central helix can play a differential role in the recognition of, or response to, CaM-binding structures.

Cofactor-induced orientation of the DNA bases in single-stranded DNA complexed with RecA protein. A fluorescence anisotropy and time-decay study.

The structure of the RecA-single-stranded DNA complex was investigated by studying the fluorescence emission of poly(deoxy-1,N6-ethenoadenylic acid (poly(d epsilon A)), a fluorescent derivative of poly(dA), under various viscosity conditions. The fluorescence intensity and average lifetime of poly(d epsilon A) are much smaller than those of nonpolymerized monoethenonucleotides (1,N6-ethenoadenosine 5'-triphosphate and 1,N6-ethenoadenine deoxyribose 5'-monophosphate) at low viscosity and reflect intramolecular base-base collisions in the polymer. They considerably increased upon RecA binding, both in the presence and absence of cofactor ATP or adenosine 5'-O-(3-thiotriphosphate). This increase, as well as the increase in fluorescence anisotropy upon RecA binding, was very similar to that which resulted from sucrose addition to free poly(d epsilon A). These observations point to a decrease in the mobility of DNA bases upon RecA binding. In the presence of cofactor, the fluorescence features became independent of viscosity. This strongly suggests the absence of base motion of significant amplitude on the time scale of the fluorescence lifetime (about 10 ns). In the absence of cofactor, however, these features remained sensitive to viscosity, implying residual local motions of the bases. Such cofactor-dependent rigid attachment of DNA bases to stiff phosphate backbone could facilitate the search for homology between two DNA molecules during recombination.

Time-resolved fluorescence study of VU-9 calmodulin, an engineered calmodulin possessing a single tryptophan residue.

An engineered calmodulin (VU-9 calmodulin), which possesses a single tryptophan residue at position 99 in calcium binding domain III, was studied by time-resolved fluorescence. At least two exponential terms are needed to describe the tryptophan fluorescence decays, either in the presence or in the absence of calcium. The characteristics of the fluorescence decays are strongly dependent upon the number of calcium ions bound per molecule of VU-9 calmodulin until half of the calcium sites are occupied, i.e., three in the absence of magnesium and two in the presence of 5 mM magnesium. A clear time-dependent spectral shift is observed in the presence of calcium. The existence of an isosbestic point in the time-resolved spectra is in agreement with a two-state model. The biexponential analysis of the 340-nm fluorescence decay during calcium titration gives parameters consistent with a two-state model in which tryptophan 99 interconverts between two different conformations, characterized by a different lifetime value, with rates altered by calcium binding. This model explains the decrease in the protein quantum yield induced by calcium binding [Kilhoffer, M. C., Roberts, D. M. Adibi, A. O., Watterson, D. M., & Haiech, J. (1989) Biochemistry (preceding paper in this issue)].

Kinetic studies of recA protein binding to a fluorescent single-stranded polynucleotide.

Fluorescence spectroscopy was used to investigate the binding of Escherichia coli recA protein to a single-stranded polynucleotide. Poly(deoxy-1,N6-ethenoadenylic acid) was prepared by reaction of chloroacetaldehyde with poly(deoxyadenylic acid). The fluorescence of poly(deoxy-1,N6-ethenoadenylic acid) was enhanced upon recA protein binding. The kinetics of the binding process were studied as a function of several parameters: ionic concentration (KCl and MgCl2), pH, nature of the nucleoside triphosphate [adenosine 5'-triphosphate or adenosine 5'-O-(gamma-thiotriphosphate)], protein and polynucleotide concentrations, polynucleotide chain length, and order of sequential additions. The observed kinetic curves exhibited a lag phase followed by a slow binding process characteristic of a nucleation-elongation mechanism with an additional slow step governing the rate of the association process. The lag phase reflecting the nucleation step was not observed when the protein was first bound to the polynucleotide before addition of adenosine 5'-triphosphate. Adenosine 5'-triphosphate induced a dissociation of the recA protein, which was immediately followed by binding of the recA-adenosine 5'-triphosphate-Mg2+ ternary complex. The origin of this "mnemonic effect" and of the different kinetic steps is discussed with respect to protein conformational changes and aggregation phenomena.

Binding of recA protein from E. coli to double-stranded DNA: influence of the degree of superhelicity.

The binding of the recA protein from E. coli to supercoiled double-stranded DNA is strongly dependent upon the superhelical density of the DNA molecule. A threshold of superhelical density is required for strong binding in the presence of ATP. This finding is consistent with a model in which recA protein first binds to unpaired regions and then polymerises on the contiguous double-stranded lattice.

Absorption and fluorescence studies of the binding of the recA gene product from E. coli to single-stranded and double-stranded DNA. Ionic strength dependence.

The binding of the recA gene product from E. coli to double-stranded and single-stranded nucleic acids has been investigated by following the change in melting temperature of duplex DNA and the fluorescence of single-stranded DNA or poly(dA) modified by reaction with chloroacetaldehyde. At low ionic strength, in the absence of Mg2+ ions, RecA protein binds preferentially to duplex DNA or poly(dA-dT). This leads to an increase of the DNA melting temperature. Stabilization of duplex DNA decreases when ionic strength or pH increases. In the presence of Mg2+ ions, preferential binding to single-stranded polynucleotides is observed. Precipitation occurs when duplex DNA begins to melt in the presence of RecA protein. From competition experiments, different single-stranded and double-stranded polydeoxynucleotides can be ranked according to their ability to bind RecA protein. Structural changes induced in nucleic acids upon RecA binding are discussed together with conformational changes induced in RecA protein upon magnesium binding.