Activation patterns of vasopressinergic and oxytocinergic brain regions following social play exposure in juvenile male and female rats.

Social play is a highly rewarding and motivated behavior predominately displayed by juveniles and expressed by nearly all mammalian species. Prior work suggested that the vasopressin (AVP) and oxytocin (OT) systems can regulate the expression of social play in sex-specific ways. Here we investigated whether there are sex differences in the recruitment of vasopressinergic and oxytocinergic brain regions following social play exposure in juvenile rats. Single-housed rats were allowed to play, in their home cage, with an age- and sex-matched unfamiliar conspecific for 10 min, or received similar handling but no partner. Double-labeled fluorescent immunohistochemistry for Fos and either AVP or OT was completed in adjacent series of tissue to determine recruitment of AVP- and OT-immunoreactive neurons in response to social play. Exposure to social play did not increase recruitment of AVP or OT neurons in the supraoptic (SO) or paraventricular (PVH) hypothalamic nuclei of either sex compared to the no-play control condition. Interestingly, there was a robust sex difference in SO recruitment, irrespective of social play condition, with males exhibiting twice the recruitment of SO-AVP and SO-OT neurons compared to females. Lastly, exposure to social play increased recruitment of the posterior bed nuclei of the stria terminalis (pBST) and the posterodorsal medial amygdalar nucleus (MEApd) compared to the no-play control condition, and this effect was most pronounced in females. Our findings revealed sex differences in the recruitment of brain regions (i) independent of play condition (i.e., SO) possibly representing a sex difference in the baseline levels of AVP and OT signaling required for typical functioning and (ii) specific to play condition (i.e., pBST, MEApd). In sum, this study provides further evidence that the neural substrates underlying social play behavior are sex-specific. This article is protected by copyright. All rights reserved.

Laboratory Studies of Nonlinear Optical Signals for Caries Detection.

Multiphoton confocal microscopy and nonlinear spectroscopy are used to investigate the caries process in dentin. Although dentin is a major calcified tissue of the teeth, its organic phase comprises type I collagen fibers. Caries drive dentin demineralization and collagen denaturation. Multiphoton microscopy is a powerful imaging technique: the biological materials are transparent to infrared frequencies and can be excited to penetration depths inaccessible to 1-photon confocal microscopy. The laser excitation greatly reduces photodamage to the sole focal region, and the signal-to-noise ratio is improved significantly. The method has been used to follow pathologic processes involving collagen fibrosis or collagen destruction based on their 2-photon excited fluorescence (2PEF) emission and second harmonic generation (SHG). Combining multiphoton imaging with nonlinear spectroscopy, we demonstrate that both 2PEF and SHG intensity of human dentin are strongly modified during the tooth caries process, and we show that the ratio between SHG and 2PEF signals is a reliable parameter to follow dental caries. The ratio of the SHG/2PEF signals measured by nonlinear optical spectroscopy provides valuable information on the caries process, specifically on the degradation of the organic matrix of dentin. The goal is to bring these nonlinear optical signals to clinical application for caries diagnosis.

Evidence of multipolar response of Bacteriorhodopsin by noncollinear second harmonic generation.

Noncollinear second harmonic generation from a Bacteriorhodopsin (BR) oriented multilayer film was systematically investigated by varying the polarization state of both fundamental beams. Both experimental results and theoretical simulations, show that the resulting polarization mapping is an useful tool to put in evidence the optical chirality of the investigated film as well as the corresponding multipolar contributions to the nonlinear.

Human dental pulp stem cells growth and osteodifferentiation on porous resorbable scaffolds.

Short Communication selected from the Oral Presentations of the 56th Congress of the Groupèment International pour la Recherche Scientifique en Stomatologie et Odontologie, Peñafiel (Portugal) May 2012.

[Not Available]. / Fonctionnalisation du Titane par des Peptides Specifiques Bi-Fonctionnels pour l'adhesion Epitheliale.

Short Communication selected from the Oral Presentations of the 56th Congress of the Groupèment International pour la Recherche Scientifique en Stomatologie et Odontologie, Peñafiel (Portugal) May 2012.

Modeling of the detachment of a molecule from a surface: illustration of the "Bell-Evans effect".

This article deals with the modeling of the detachment of a molecule initially adsorbed on a surface and submitted to an external force whose strength increases with time. By means of an atomic force microscope (AFM), it is possible to measure the force when the molecule separates from the substrate. However, it is known that this force depends to a large extend on the rate at which the pulling force is applied ("Bell-Evans effect"). Two models are described to illustrate this behavior. First, a random walk approach is suggested to reveal the fundamental principle of the escape over a time-dependent energy barrier. Second, a multi bead-and-spring model is proposed to mimic the AFM experiment and numerical simulations, based on Brownian dynamics, are performed.

Semi-automatized processing of AFM force-spectroscopy data.

Atomic force microscopy operated in the force-spectroscopy mode is now a widespread technique, often used to investigate ligand-receptor interactions with the goal of measuring forces at the individual molecule level. However, in an experiment, the simultaneous interaction of several ligand/receptor pairs cannot be excluded. This may produce complicated force curves, although unambiguous ruptures are sometimes observed. In the case of the non-specific adhesion of molecules, such as fibrinogen, to a surface, it is usually difficult to identify the real events on the force curves. This can render the application of fixed rules uneasy and in addition can introduce some degree of arbitrariness if the analysis has to be performed by hand. In the present paper a computer algorithm, aimed at speeding up the processing, and at applying selection rules in a reproducible manner, is proposed. It is applied to force recordings performed at various retraction velocities, thus various loading rates. The influence on the evaluation of the rupture forces of the different parameters that can be set by the operator is discussed.

Unbinding process of adsorbed proteins under external stress studied by atomic force microscopy spectroscopy.

We report the study of the dynamics of the unbinding process under a force load f of adsorbed proteins (fibrinogen) on a solid surface (hydrophilic silica) by means of atomic force microscopy spectroscopy. By varying the loading rate r(f), defined by f = r(f) t, t being the time, we find that, as for specific interactions, the mean rupture force increases with r(f). This unbinding process is analyzed in the framework of the widely used Bell model. The typical dissociation rate at zero force entering in the model lies between 0. 02 and 0.6 s(-1). Each measured rupture is characterized by a force f(0), which appears to be quantized in integer multiples of 180-200 pN.

Protein interactions with polyelectrolyte multilayers: interactions between human serum albumin and polystyrene sulfonate/polyallylamine multilayers.

The interactions between polystyrenesulfonate (PSS)/polyallylamine (PAH) multilayers with human serum albumin (HSA) were investigated by means of scanning angle reflectometry (SAR). We find that albumin adsorbs both on multilayers terminating with PSS (negatively charged) or PAH (positively charged) polyelectrolytes. On films terminating with PSS only, an albumin equivalent monolayer is found whereas when PAH constitutes the outer layer, albumin interacts with the multilayer in such a way as to form a protein film that extends over thicknesses that can be as high as four times the largest dimension of the native albumin molecule. Once the protein film is formed, it is found that when the albumin solution is replaced by a pure buffer solution of same ionic strength as the adsorption solution almost no desorption takes place. On the other hand, when a buffer solution of higher ionic strength is brought in contact with the albumin film, a significant amount of adsorbed proteins is released. One also observes that, for albumin solutions of a given protein concentration, the adsorbed protein amount depends on the ionic strength of the adsorption solution. On surfaces terminating with PAH, the adsorbed protein amount first increases rapidly but passes through a maximum and decreases with the ionic strength. The ionic strength corresponding to the maximum of the adsorbed albumin amount itself depends on the albumin concentration. On the other hand, on films terminating with PSS the adsorbed amount increases with the salt concentration before leveling-off. These results show that the underlying complexity of concentration and pH dependent adsorption/desorption equilibria often simply termed "protein adsorption" is the result of antagonist competing interactions that are mainly of electrostatic origin. We also propose two microscopic models, that are compatible with our experimental observations.

Kinetic and thermodynamic study of the bacteriorhodopsin photocycle over a wide pH range.

The photocycle of bacteriorhodopsin and its thermodynamic parameters were studied in the pH range of 4.5-9. Measurements were performed at five different wavelengths (410, 500, 570, 610, and 650 nm), in the time interval 300 ns to 0.5 s, at six temperatures between 5 and 30 degreesC. Data were fitted to different photocycle models. The sequential model with reversible reactions gave a good fit, and the linear character of the Eyring plots was fulfilled. The parallel model with unidirectional reactions gave a poor fit, and the Eyring plot of the rate constants did not follow the expected linear behavior. When a parallel model with reversible reactions, which has twice as many free parameters as the sequential model, was considered, the quality of the fit did not improve and the Eyring plots were not linear. The sequential model was used to determine the thermodynamic activation parameters (activation enthalpy, entropy, and free energy) of the transitions and the free energy levels of the intermediates. pH dependence of the parameters revealed details of the transitions between the intermediates: the transitions M1 to M2 and N to O disclosed a large entropy increase, which could be interpreted as a loosening of the protein structure. The pH dependence of the energy levels explains the disappearance of intermediate O at high pH. A hypothesis is proposed to interpret the relation between the observed pKa of the photocycle energetics and the role of several amino acids in the protein.

Electric signals during the bacteriorhodopsin photocycle, determined over a wide pH range.

From the electric signals measured after photoexcitation, the electrogenicity of the photocycle intermediates of bacteriorhodopsin were determined in a pH range of 4.5-9. Current measurements and absorption kinetic signals at five wavelengths were recorded in the time interval from 300 ns to 0.5 s. To fit the data, the model containing sequential intermediates connected by reversible first-order reactions was used. The electrogenicities were calculated from the integral of the current signal, by using the time-dependent concentrations of the intermediates, obtained from the fits. Almost all of the calculated electrogenicities were pH independent, suggesting that the charge motions occur inside the protein. Only the N intermediate exhibited pH-dependent electrogenicity, implying that the protonation of Asp96, from the intracellular part of the protein, is not from a well-determined proton donor. The calculated electrogenicities gave good approximations of all of the details of the measured electric signals.

The role of water in the extracellular half channel of bacteriorhodopsin.

The changes in the photocycle of the wild type and several mutant bacteriorhodopsin (D96N, E204Q, and D212N) were studied on dried samples, at relative humidities of 100% and 50%. Samples were prepared from suspensions at pH approximately 5 and at pH approximately 9. Intermediate M with unprotonated Schiff base was observed at the lower humidity, even in the case where the photocycle in suspension did not contain this intermediate (mutant D212N, high pH). The photocycle of the dried sample stopped at intermediate M1 in the extracellular conformation; conformation change, switching the accessibility of the Schiff base to the cytoplasmic side, and proton transport did not occur. The photocycle decayed slowly by dissipating the absorbed energy of the photon, and the protein returned to its initial bacteriorhodopsin state, through several M1-like substates. These substates presumably reflect different paths of the proton back to the Schiff base, as a consequence of the bacteriorhodopsin adopting different conformations by stiffening on dehydration. All intermediates requiring conformational change were hindered in the dried form. The concentration of intermediate L, which appears after isomerization of the retinal from all-trans to 13-cis, during local relaxation of the protein, was unusually low in dried samples. The lack of intermediates N and O demonstrated that the M state did not undergo a change from the extracellular to the cytoplasmic conformation (M1 to M2 transition), as already indicated by Fourier transform infrared spectroscopy, quasielastic incoherent neutron scattering, and electric signal measurements described in the literature.

Combined optical and photoelectric study of the photocycle of 13-cis bacteriorhodopsin.

The photocycle of the 13-cis retinal containing bacteriorhodopsin was studied by three different techniques. The optical multichannel analyzer monitored the spectral changes during the photocycle and gave information about the number and the spectrum of the intermediates. The absorption kinetic measurements provided the possibility of following the absorbance changes at several characteristic wavelengths. The electric signal provided information about the charge motions during the photocycle. The results reveal the existence of two intermediates in the 13-cis photocycle, one with a short lifetime having an average of 1.7 microseconds and an absorption maximum at 620 nm. The other, a long-living intermediate, has a lifetime of about 50 ms and an absorption maximum around 585 nm. The data analysis suggests that these intermediates are in two parallel branches of the photocycle, and branching from the intermediate with the shorter lifetime might be responsible for the light-adaptation process.

Study of the photocycle and charge motions of the bacteriorhodopsin mutant D96N.

Absorption kinetic and electric measurements were performed on oriented purple membranes of D96N bacteriorhodopsin mutant embedded in polyacrylamide gel and the kinetic parameters of the photointermediates determined. The rate constants, obtained from fits to time-dependent concentrations, were used to calculate the relative electrogenicity of the intermediates. The signals were analyzed on the basis of different photocycle models. The preferred model is the sequential one with reversible reaction. To improve the quality of the fits the necessity of introducing a second L intermediate arose. We also attempted to interpret our data in the view of reversible reactions containing two parallel photocycles, but the pH dependencies of the rate constants and electrogenicities favored the model containing sequential reversible transitions. A fast equilibrium for the L2<==>M1 transition and a strong pH dependence of the M2 electrogenicity was found, indicating that the M1 to M2 transition involves complex charge motions, as is expected in a conformational change of the protein.