Peritoneal fluid modifies the response of human spermatozoa to follicular fluid

The aim of this study was to elucidate the mechanism involved in the acrosome reaction (AR) induced by follicular fluid (FF) in spermatozoa previously exposed to peritoneal fluid (PF). The influence of progesterone was also investigated. Semen samples were from 18 normozoospermic donors. PF samples were from 13 women with unexplained infertility and from a woman treated with synthetic progestagen. FF samples were collected from six women undergoing IVF/embryo transfer and pooled. Motile spermatozoa were capacitated overnight and a kinetic and inhibition study on the FF-induced AR was performed. Spermatozoa pretreated with PF were challenged with either FF or progesterone. The ability of progesterone- and progestagen-supplemented PF to induce AR was analysed. Enzyme-digested PF was also tested. Pre-incubation with PF for 60 min completely prevented the FF-induced AR; spermatozoa treated with PF were unable to respond to FF or progesterone and this effect was not reversible. Progesterone- and progestagen-supplemented PF stimulated the AR relative to controls. Enzyme-digested PF did not have an inhibitory capacity. These data strongly suggest that there are one or more inhibitory proteins in PF that interact with spermatozoa so as to prevent access of progesterone to its receptor and thus inhibit the occurrence of the AR. The oviduct, or Fallopian tube, provides a place for spermatozoa and egg transport and storage, fertilization and early embryo development. If ovulation has not occurred, spermatozoa may reside in the oviduct for several hours or even a few days, awaiting oocyte arrival. It is assumed that fluids present in the female genital tract may have a role in synchronizing the timing required to guarantee the success of fertilization. We previously observed that the peritoneal fluid that bathes the peritoneal cavity is a suitable medium for sperm survival and we also reported that this fluid could stabilize spermatozoa. In this study we show further evidence that the exposure to peritoneal fluid modifies the response of spermatozoa to oocyte signals.Fil: Caille, Adriana M.. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Berta, Cesar L.. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Cuasnicu, Patricia Sara. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Munuce, María J.. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentin

Participatory scenario development for integrated assessment of nutrient flows in a Catalan river catchment

Rivers in developed regions are under significant stress due to nutrient enrichment generated mainly by human activities. Excess nitrogen and phosphorus emissions are the product of complex dynamic systems influenced by various factors such as demographic, socio-economic and technological development. Using a Catalan river catchment, La Tordera (North-East of Spain), as a case study of an integrated and interdisciplinary environmental assessment of nutrient flows, we present and discuss the development of narrative socio-economic scenarios through a participatory process for the sustainable management of the anthropogenic sources of nutrients, nitrogen and phosphorus. In this context, scenarios are an appropriate tool to assist nutrient emissions modelling, and to assess impacts, possible pathways for socio-economic development and associated uncertainties. Evaluated against the 1993–2003 baseline period, scenarios target the 2030 horizon, i.e. through the implementation process of the Water Framework Directive (Directive 2000/60/EC). After a critical examination of the methodology used in the participatory development of socio-economic scenarios, we present four possible futures (or perspectives) for the Catalan river catchment conceived by stakeholders invited to a workshop. Keys to the success of such a participatory process were trust, which enhanced openness, and disagreements, which fostered the group's creativity for scenario development. The translation of narrative socio-economic scenarios into meaningful nutrient emission scenarios is also discussed. By integrating findings of natural sciences and socio-economic analysis, we aim to assist decision makers and stakeholders in evaluating optimal management strategies for the anthropogenic sources of nitrogen and phosphorus

Straight GDP-Tubulin Protofilaments Form in the Presence of Taxol

International audienceMicrotubules exist in dynamic equilibrium, growing and shrinking by the addition or loss of tubulin dimers from the ends of protofilaments. The hydrolysis of GTP in b-tubulin destabilizes the microtubule lattice by increasing the curvature of protofilaments in the microtubule and putting strain on the lattice. The ob- servation that protofilament curvature depends on GTP hydrolysis suggests that microtubule destabil- izers and stabilizers work by modulating the curvature of the microtubule lattice itself. Indeed, the microtu- bule destabilizer MCAK has been shown to increase the curvature of protofilaments during depolymeriza- tion. Here, we show that the atomic force microscopy (AFM) of individual tubulin protofilaments provides sufficient resolution to allow the imaging of single pro- tofilaments in their native environment. By using this assay, we confirm previous results for the effects of GTP hydrolysis and MCAK on the conformation of pro- tofilaments. We go on to show that taxol stabilizes microtubules by straightening the GDP protofilament and slowing down the transition of protofilaments from straight to a curved configuration

Simulations of a single membrane between two walls using a Monte Carlo method

Quantitative theory of interbilayer interactions is essential to interpret x-ray scattering data and to elucidate these interactions for biologically relevant systems. For this purpose Monte Carlo simulations have been performed to obtain pressure P and positional fluctuations sigma. A new method, called Fourier Monte-Carlo (FMC), that is based on a Fourier representation of the displacement field, is developed and its superiority over the standard method is demonstrated. The FMC method is applied to simulating a single membrane between two hard walls, which models a stack of lipid bilayer membranes with non-harmonic interactions. Finite size scaling is demonstrated and used to obtain accurate values for P and sigma in the limit of a large continuous membrane. The results are compared with perturbation theory approximations, and numerical differences are found in the non-harmonic case. Therefore, the FMC method, rather than the approximations, should be used for establishing the connection between model potentials and observable quantities, as well as for pure modeling purposes.Comment: 10 pages, 10 figure

Consortin, a trans-Golgi network cargo receptor for the plasma membrane targeting and recycling of connexins

Targeting of numerous transmembrane proteins to the cell surface is thought to depend on their recognition by cargo receptors that interact with the adaptor machinery for anterograde traffic at the distal end of the Golgi complex. We report here on consortin, a novel integral membrane protein that is predicted to be intrinsically disordered, i.e. that contains large segments whose native state is unstructured. We identified consortin as a binding partner of connexins, the building blocks of gap junctions. Consortin is located at the trans-Golgi network (TGN), in tubulovesicular transport organelles, and at the plasma membrane. It directly interacts with the TGN clathrin adaptors GGA1 and GGA2, and disruption of this interaction by expression of a consortin mutant lacking the acidic cluster-dileucine (DXXLL) GGA interaction motif causes an intracellular accumulation of several connexins. RNA interference-mediated silencing of consortin expression in HeLa cells blocks the cell surface targeting of these connexins, which accumulate intracellularly, whereas partial depletion and redistribution of the consortin pool slows down the intracellular degradation of gap junction plaques. Altogether, our results show that, by studying connexin trafficking, we have identified the first TGN cargo receptor for the targeting of transmembrane proteins to the plasma membrane. The identification of consortin provides in addition a potential target for therapies aimed at diseases in which connexin traffic is altered, including cardiac ischemia, peripheral neuropathies, cataracts and hearing impairment. Sequence accession numbers. GenBank: Human CNST cDNA, NM_152609; mouse Cnst cDNA, NM_14610

Functional significance of repressor element 1 silencing transcription factor (REST) target genes in pancreatic beta cells

Aims/hypothesis: The expression of several neuronal genes in pancreatic beta cells is due to the absence of the transcription factor repressor element 1 (RE-1) silencing transcription factor (REST). The identification of these traits and their functional significance in beta cells has only been partly elucidated. Herein, we investigated the biological consequences of a repression of REST target genes by expressing REST in beta cells. Methods: The effect of REST expression on glucose homeostasis, insulin content and release, and beta cell mass was analysed in transgenic mice selectively expressing REST in beta cells. Relevant target genes were identified in INS-1E and primary beta cells expressing REST. Results: Transgenic mice featuring a beta cell-targeted expression of REST exhibited glucose intolerance and reduced beta cell mass. In primary beta cells, REST repressed several proteins of the exocytotic machinery, including synaptosomal-associated protein (SNAP) 25, synaptotagmin (SYT) IV, SYT VII, SYT IX and complexin II; it impaired first and second phases of insulin secretion. Using RNA interference in INS-1E cells, we showed that SYT IV and SYT VII were implicated in the control of insulin release. Conclusions/interpretation: The data document the critical role of REST target genes in pancreatic beta cells. Specifically, we provide evidence that the downregulation of these genes is detrimental for the exocytosis of large dense core vesicles, thus contributing to beta cell dysfunction and impaired glucose homeostasi

Coulombian Disorder in Periodic Systems

We study the effect of unscreened charged impurities on periodic systems. We show that the long wavelength component of the disorder becomes long ranged and dominates static correlation functions. On the other hand, because of the statistical tilt symmetry, dynamical properties such as pinning remain unaffected. As a concrete example, we focus on the effect of Coulombian disorder generated by charged impurities, on 3D charge density waves with non local elasticity. We calculate the x-ray intensity and find that it is identical to the one produced by thermal fluctuations in a disorder-free smectic-A. We discuss the consequences of these results for experiments.Comment: 11 pages, 3 figures, revtex

Template-free 13-protofilament microtubule–MAP assembly visualized at 8 A resolution

Microtubule-associated proteins (MAPs) are essential for regulating and organizing cellular microtubules (MTs). However, our mechanistic understanding of MAP function is limited by a lack of detailed structural information. Using cryo-electron microscopy and single particle algorithms, we solved the 8 Å structure of doublecortin (DCX)-stabilized MTs. Because of DCX’s unusual ability to specifically nucleate and stabilize 13-protofilament MTs, our reconstruction provides unprecedented insight into the structure of MTs with an in vivo architecture, and in the absence of a stabilizing drug. DCX specifically recognizes the corner of four tubulin dimers, a binding mode ideally suited to stabilizing both lateral and longitudinal lattice contacts. A striking consequence of this is that DCX does not bind the MT seam. DCX binding on the MT surface indirectly stabilizes conserved tubulin–tubulin lateral contacts in the MT lumen, operating independently of the nucleotide bound to tubulin. DCX’s exquisite binding selectivity uncovers important insights into regulation of cellular MTs