Single-molecule analysis of dynamics and interactions of the SecYEG translocon.

Protein translocation and insertion into the bacterial cytoplasmic membrane are the essential processes mediated by the Sec machinery. The core machinery is composed of the membrane-embedded translocon SecYEG that interacts with the secretion-dedicated ATPase SecA and translating ribosomes. Despite the simplicity and the available structural insights on the system, diverse molecular mechanisms and functional dynamics have been proposed. Here, we employ total internal reflection fluorescence microscopy to study the oligomeric state and diffusion of SecYEG translocons in supported lipid bilayers at the single-molecule level. Silane-based coating ensured the mobility of lipids and reconstituted translocons within the bilayer. Brightness analysis suggested that approx. 70% of the translocons were monomeric. The translocons remained in a monomeric form upon ribosome binding, but partial oligomerization occurred in the presence of nucleotide-free SecA. Individual trajectories of SecYEG in the lipid bilayer revealed dynamic heterogeneity of diffusion, as translocons commonly switched between slow and fast mobility modes with corresponding diffusion coefficients of 0.03 and 0.7 µm2 ·s-1 . Interactions with SecA ATPase had a minor effect on the lateral mobility, while bound ribosome:nascent chain complexes substantially hindered the diffusion of single translocons. Notably, the mobility of the translocon:ribosome complexes was not affected by the solvent viscosity or macromolecular crowding modulated by Ficoll PM 70, so it was largely determined by interactions within the lipid bilayer and at the interface. We suggest that the complex mobility of SecYEG arises from the conformational dynamics of the translocon and protein:lipid interactions.

Two distinct anionic phospholipid-dependent events involved in SecA-mediated protein translocation.

Protein translocation across the bacterial cytoplasmic membrane is an essential process catalyzed by the Sec translocase, which in its minimal form consists of the protein-conducting channel SecYEG, and the motor ATPase SecA. SecA binds via its positively charged N-terminus to membranes containing anionic phospholipids, leading to a lipid-bound intermediate. This interaction induces a conformational change in SecA, resulting in a high-affinity association with SecYEG, which initiates protein translocation. Here, we examined the effect of anionic lipids on the SecA-SecYEG interaction in more detail, and discovered a second, yet unknown, anionic lipid-dependent event that stimulates protein translocation. Based on molecular dynamics simulations we identified an anionic lipid-enriched region in vicinity of the lateral gate of SecY. Here, the anionic lipid headgroup accesses the lateral gate, thereby stabilizing the pre-open state of the channel. The simulations suggest flip-flop movement of phospholipid along the lateral gate. Electrostatic contribution of the anionic phospholipids at the lateral gate may directly stabilize positively charged residues of the signal sequence of an incoming preprotein. Such a mechanism allows for the correct positioning of the entrant peptide, thereby providing a long-sought explanation for the role of anionic lipids in signal sequence folding during protein translocation.

Effects of male postpartum depression on father-infant interaction: The mediating role of face processing.

It is estimated that postpartum depression affects up to 25% of men. Despite such high prevalence, the majority of studies on postpartum depression are focused on mothers, and the role of paternal depression and its effects on infant development have been overlooked by researchers and clinicians. The present study aimed to fill this gap by investigating the effect of paternal postpartum depression on father-infant interactions. In addition, we examined whether differences in face recognition mediated the effects of paternal postpartum depression on father-infant interactions. A total of 61 father-infant dyads (17 postpartum depression, 44 controls) took part in the study. Results revealed that compared to controls, fathers with postpartum depression had a worse pattern of interaction with their infants on measures of responsiveness, mood, and sensitivity; they also had greater difficulty in recognizing happy adult faces, but greater facility in recognizing sad adult faces. Depressed fathers attributed greater intensities to sad adult and infant faces. The tendency to attribute greater intensity to sad adult faces was confirmed as a partial mediator of the effect of paternal postpartum depression on measures of father responsiveness and as a full mediator of the effects of paternal depression on father sensitivity. Clinical implications and suggestions for further studies are discussed.

Se estima que la depresión posterior al parto afecta hasta un 25% de los hombres. A pesar de tan alta prevalencia, la mayoría de los estudios sobre la depresión posterior al parto se enfocan en las madres, y los investigadores y clínicos han pasado por alto el papel de la depresión paterna y sus efectos en el desarrollo del infante. El presente estudio se propuso llenar ese vacío investigando el efecto que la depresión paterna posterior al parto tiene en las interacciones papá-infante. Adicionalmente, examinamos si las diferencias en reconocer las caras mediaron los efectos que la depresión paterna posterior al parto tiene en las interacciones papá-infante. Sesenta y una díadas papá-infante (17 en el grupo de depresión posterior al parto, 44 en el grupo de control) participaron en el estudio. Los resultados revelaron que, comparados con el grupo de control, los papás con depresión posterior al parto presentaban un peor patrón de interacción con sus infantes en medidas de capacidad de respuesta, estado de ánimo y sensibilidad; ellos también tuvieron mayores dificultades en reconocer caras adultas felices, pero con mayor facilidad reconocieron caras adultas tristes. Los padres deprimidos atribuyeron una mayor intensidad a las caras tristes de adultos e infantes. Se confirmó la tendencia de atribuir una mayor intensidad a las caras adultas tristes como un mediador parcial del efecto que la depresión paterna posterior al parto tiene sobre la calidad de respuesta del papá y como un completo mediador de los efectos que la depresión paterna tiene sobre la sensibilidad del papá. Se discuten las implicaciones y sugerencias clínicas para futuros estudios.

On estime que la dépression postpartum affect jusqu'à 25% des hommes. En dépit d'une telle prévalence élevée la majorité des études sur la dépression postpartum porte sur les mères et le rôle de la dépression paternelle et de ses effets sur le développement du nourrisson a été négligé par les chercheurs et les cliniciens. Cette étude s'est donné pour but de remplir ce fossé en recherchant l'effet de la dépression postpartum sur les interactions père-nourrisson. De plus, nous avons examiné si les différences dans la reconnaissance faciale ont assuré la médiation des effets de la dépression paternelle postpartum sur les interactions père-nourrisson. 61 dyades père-bébé (17 dépression postpartum, 44 contrôles) ont pris part à l'étude. Les résultats ont révélé que, comparés aux contrôles, les pères avec la dépression postpartum faisaient état d'un pattern d'interaction avec leur bébé pire sur les mesures de réactivité, d'humeur et de sensibilité. Ils avaient aussi plus de difficulté à reconnaître les visages adultes heureux mais une plus grande facilité à reconnaître les visages adultes tristes. Les pères déprimés ont attribué de plus grandes intensités aux visages tristes de l'adulte et du bébé. La tendance à attribuer une plus grande intensité aux visages tristes de l'adulte a été confirmée comme un médiateur partiel de l'effet de la dépression paternelle postpartum sur la réaction du père et comme un médiateur total sur les effets de la dépression paternelle sur la sensibilité du père. Les implications cliniques et des suggestions de recherche sont discutées.

Does winter desiccation account for seasonal increases in supercooling capacity of Norway spruce bud primordia?

Bud primordia of Picea abies (L.) H. Karst. remain ice free at subzero temperatures by supercooling. Once ice forms inside the primordium, it is immediately injured. Supercooling capacity increases seasonally from ~-5 °C to as much as -50 °C by currently unknown mechanisms. Among other prerequisites, dehydration of tissues over the winter months has been considered to play a key role in freezing tolerance. In this regard, the water content of bud primordia may be crucial, especially in reference to supercooling. In order to assess the role of dehydration in supercooling capacity, seasonal changes in supercooling capacity and the water potential of bud primordia of Picea abies (L.) H. Karst were measured at two sites that differed by 1298 m in elevation, after artificial frost hardening and dehardening treatments and after controlled bench drying. The extent of supercooling of bud primordia varied from -7 °C in summer to -24.6 °C in winter, a difference of 17.6 -19.3 K. Total actual water potential (Ψtact) of bud primordia was -2 MPa in summer and decreased to a mean of -3.8 MPa in midwinter. The decline involved dehydration, and to a lesser extent, osmoregulation. At decreased Ψtact values (<3.0 MPa), supercooling capacity significantly increased <-19.5 °C, however, the correlation between actual water potential and supercooling capacity was poor. Frost-hardening treatments increased the supercooling capacity of bud primordia (-0.6 K day-1) and lowered Ψtact (-0.2 MPa day-1). Frost-dehardening treatments reduced supercooling capacity (+1.1 K day-1), and at the same time, increased Ψtact (+0.3 MPa day-1). In contrast, artificial drying of bud primordia in the range observed seasonally (-2.0 MPa) had no effect on supercooling capacity. These results suggest that there is no causal relationship between desiccation and the supercooling capacity of bud primordia in P. abies, but rather it involves other compounds within the cells of the bud primordium that reduce the water potential.

Lipids Activate SecA for High Affinity Binding to the SecYEG Complex.

Protein translocation across the bacterial cytoplasmic membrane is an essential process catalyzed predominantly by the Sec translocase. This system consists of the membrane-embedded protein-conducting channel SecYEG, the motor ATPase SecA, and the heterotrimeric SecDFyajC membrane protein complex. Previous studies suggest that anionic lipids are essential for SecA activity and that the N terminus of SecA is capable of penetrating the lipid bilayer. The role of lipid binding, however, has remained elusive. By employing differently sized nanodiscs reconstituted with single SecYEG complexes and comprising varying amounts of lipids, we establish that SecA gains access to the SecYEG complex via a lipid-bound intermediate state, whereas acidic phospholipids allosterically activate SecA for ATP-dependent protein translocation.

Transfemoral aortic valve implantation with the repositionable Lotus valve compared with the balloon-expandable Edwards Sapien 3 valve.

BACKGROUND: The rate of paravalvular aortic insufficiency (AI) with transcatheter aortic valve implantation (TAVI) with first generation devices was higher compared with surgical replacement. Residual AI after TAVI has been linked to an increased mortality rate. We compared two second generation TAVI devices - the repositionable Lotus valve with the balloon-expandable Edwards Sapien 3 valve - regarding procedural and 30 day outcome. METHODS AND RESULTS: In 78 patients with severe aortic stenosis undergoing transfemoral TAVI we evaluated post-procedural paravalvular AI, device success and early safety according to VARC criteria. Valve size was based on a 256-multislice computed tomography. Patients were followed for 30 days. The Lotus valve (N = 26) and the Edwards Sapien 3 valve (N = 52) were implanted under fluoroscopic guidance. Baseline characteristics were similar between groups. Perimeter derived annulus diameter did not differ with 25.7 ± 1.6mm for Lotus and 25.2 ± 2.1mm for Edwards Sapien 3 patients. After TAVI aortography and transthoracic echocardiography revealed no moderate or severe AI. The rate of mild AI was 12% for Lotus and 15% for Edwards Sapien 3 (p = 0.62). There were no deaths, stroke, annulus rupture or coronary obstruction. Device success was 96% and 98% (p = 0.61), early safety according to VARC 11.5% in both groups (p = 1.0) and the need for pacemaker implantation 27% and 4% (p < 0.003), respectively. CONCLUSIONS: TAVI with second-generation devices was associated with no moderate or severe AI and a low rate of mild AI. Device success was high for Lotus and Edwards Sapien 3 while the need for permanent pacemaker was significantly higher with the Lotus valve.

ToF-SIMS measurements with topographic information in combined images.

In 2D and 3D time-of-flight secondary ion mass spectrometric (ToF-SIMS) analysis, accentuated structures on the sample surface induce distorted element distributions in the measurement. The origin of this effect is the 45° incidence angle of the analysis beam, recording planar images with distortion of the sample surface. For the generation of correct element distributions, these artifacts associated with the sample surface need to be eliminated by measuring the sample surface topography and applying suitable algorithms. For this purpose, the next generation of ToF-SIMS instruments will feature a scanning probe microscope directly implemented in the sample chamber which allows the performance of topography measurements in situ. This work presents the combination of 2D and 3D ToF-SIMS analysis with topographic measurements by ex situ techniques such as atomic force microscopy (AFM), confocal microscopy (CM), and digital holographic microscopy (DHM). The concept of the combination of topographic and ToF-SIMS measurements in a single representation was applied to organic and inorganic samples featuring surface structures in the nanometer and micrometer ranges. The correct representation of planar and distorted ToF-SIMS images was achieved by the combination of topographic data with images of 2D as well as 3D ToF-SIMS measurements, using either AFM, CM, or DHM for the recording of topographic data.

Escherichia coli TatA and TatB proteins have N-out, C-in topology in intact cells.

The twin arginine protein transport (Tat) system translocates folded proteins across the cytoplasmic membrane of prokaryotes and the thylakoid membrane of chloroplasts. In Escherichia coli, TatA, TatB, and TatC are essential components of the machinery. A complex of TatB and TatC acts as the substrate receptor, whereas TatA is proposed to form the Tat transport channel. TatA and TatB are related proteins that comprise an N-terminal transmembrane helix and an adjacent amphipathic helix. Previous studies addressing the topological organization of TatA have given conflicting results. In this study, we have addressed the topological arrangement of TatA and TatB in intact cells by labeling of engineered cysteine residues with the membrane-impermeable thiol reagent methoxypolyethylene glycol maleimide. Our results show that TatA and TatB share an N-out, C-in topology, with no evidence that the amphipathic helices of either protein are exposed at the periplasmic side of the membrane. We further show that the N-out, C-in topology of TatA is fixed and is not affected by the absence of other Tat components or by the overproduction of a Tat substrate. These data indicate that topological reorganization of TatA is unlikely to accompany Tat-dependent protein transport.

Serotonin-induced decrease of intracellular Ca(2+) release in platelets of bulimic patients normalizes during treatment.

Numerous symptoms related to eating disorders have been shown to be influenced by serotonergic (5-HT) functioning, with the 5-HT(2A) receptor subtype being one of the most relevant involved in the pathophysiology of bulimia nervosa (BN). In line with this, Ca(2+) mobilization as mediated by 5-HT(2) receptors in platelets was shown to serve as a peripheral model for central nervous 5-HT functioning. Here, the 5-HT-induced intracellular Ca(2+) mobilization in platelets was measured in 13 female normal weight bulimic patients (14-18 years) upon admission and at the end of inpatient treatment. Findings were compared to 21 age-matched healthy female adolescents. 5-HT-induced Ca(2+) release was significantly decreased in bulimic patients upon admission and normalized during inpatient treatment. Antidepressive medication caused a significant improvement. The data provide further evidence that altered 5-HT(2) receptor functioning is involved in the pathophysiological underpinnings in BN.

KlRHO1 and KlPKC1 are essential for cell integrity signalling in Kluyveromyces lactis.

Cell integrity in yeasts is ensured by a rigid cell wall whose synthesis is triggered by a MAP kinase-mediated signal-transduction cascade. Upstream regulatory components of this pathway in Saccharomyces cerevisiae involve a single protein kinase C, which is regulated by interaction with the small GTPase Rho1. Here, two genes were isolated which encode these proteins from Kluyveromyces lactis (KlPKC1 and KlRHO1). Sequencing showed ORFs which encode proteins of 1,161 and 208 amino acids, respectively. The deduced proteins shared 59 and 85 % overall amino acid identities, respectively, with their homologues from S. cerevisiae. Null mutants in both genes were non-viable, as shown by tetrad analyses of the heterozygous diploid strains. Overexpression of the KlRHO1 gene under the control of the ScGAL1 promoter severely impaired growth in both S. cerevisiae and K. lactis. On the other hand, a similar construct with KlPKC1 did not show a pronounced phenotype. Two-hybrid analyses showed interaction between Rho1 and Pkc1 for the K. lactis proteins and their S. cerevisiae homologues. A green fluorescent protein (GFP) fusion to the C-terminal end of KlPkc1 located the protein to patches in the growing bud, and at certain stages of the division process also to the bud neck. N-terminal GFP fusions to KlRho1 localized mainly to the cell surface (presumably the cytoplasmic side of the plasma membrane) and to the vacuole, with some indications of traffic from the former to the latter. Thus, KlPkc1 and KlRho1 have been shown to serve vital functions in K. lactis, to interact in cell integrity signalling and to traffic between the plasma membrane and the vacuole.

Strain-specific responses of inbred mice to ethanol following food shortage.

Specific inbred mouse strains such as C57BL/6J and DBA/2J show differences in consumption of and reaction on drugs of abuse. For example, C57BL/6J mice voluntarily consume greater amounts of ethanol than DBA/2J mice. Recently, it could be shown that a short environmental experience--12 days of food shortage followed by a recovery period--has a strong impact on strain-specific reactions to amphetamine. The purpose of the present study was to examine whether food shortage experience has an effect on ethanol responses. The effect of a period of 12 days food restriction which resulted in a weight loss of 20% body weight and which was followed by a complete recovery period was studied on ethanol self-administration and ethanol-induced locomotor activity in C57BL/6Ico and DBA/2Ico inbred mouse strains. The experience of food shortage led to a higher ethanol intake and preference in C57BL/6Ico mice compared to control animals without food shortage experience. In contrast DBA/2Ico showed no difference in ethanol intake or preference following this experience. The effect of ethanol onto locomotor activity of both mice strains was affected only in the case of DBA/2Ico mice, where food shortage experience resulted in a significantly higher ethanol-induced locomotor activity. The present data show that in inbred mouse strains environmental experiences can have a strong impact onto the effects of ethanol. In conclusion, in the field of preclinical alcohol research gene x environment interactions in specific inbred mouse strains can contribute strongly to the outcome of studies and more specifically food shortage can profoundly affect the outcome of alcohol studies in mice.

mGluR5 antagonist MPEP reduces ethanol-seeking and relapse behavior.

The glutamatergic system plays an important role in mediating neurobehavioral effects of ethanol. Metabotropic glutamate receptors subtype 5 (mGluR5) are modulators of glutamatergic neurotransmission and are abundant in brain regions known to be involved in ethanol self-administration. Here, we studied the effects of 2-methyl-6-(phenylethynyl)-pyridine (MPEP), a highly potent, noncompetitive mGlu5 receptor antagonist, on voluntary ethanol consumption and relapse behavior. For this purpose, we used two models for the measurement of relapse behavior: (i) reinstatement of ethanol-seeking behavior by drug-associated cues and (ii) the alcohol deprivation effect in long-term ethanol-consuming rats. In the first set of experiments, rats were trained to lever press for ethanol in the presence of a distinct set of cues. After extinction, the animals were exposed to the respective cues that initiated reinstatement of responding. A response-contingent ethanol prime further enhanced responding compared to the conditioned cues alone. Under these conditions, MPEP (0, 1, 3, and 10 mg/kg) attenuated ethanol seeking significantly and in a dose-related manner. However, at the highest dose, MPEP also decreased the number of inactive lever responses. In the second set of experiments, rats with 1 year of ethanol experience and repeated deprivation phases were used. A subchronic treatment with MPEP (twice daily; 0, 3, and 10 mg/kg) resulted in a significant and dose-dependent reduction of the alcohol deprivation effect (ADE). Although the same MPEP treatment regimen decreased baseline drinking, this effect was not as pronounced as on the ADE. These results show in two commonly used models of relapse to ethanol that pharmacological targeting of mGlu5 receptors may be a promising approach for the treatment of alcoholism.