Synaptosome-associated protein of 25 kilodaltons in oocytes and steroid-producing cells of rat and human ovary: molecular analysis and regulation by gonadotropins

The synaptosome-associated protein of 25 kDa (SNAP-25) is crucially involved in exocytosis in neurons. The aim of this study was to investigate whether it is present in the ovary. We found SNAP-25 to be expressed in nonneuronal cells of the rat and human ovary, namely in all oocytes and in steroidogenic cells, including granulosa cells (GC) of large antral follicles and luteal cells. Both isoforms, SNAP-25a and b, were found in the ovary. Oocytes obtained by laser capture microdissection were shown to express SNAP-25b, whereas SNAP-25a was found in rat GC and human luteinized GC. Immunohistochemical observations of strong SNAP-25 staining in GC of large growing antral follicles compared with absent or weak staining in small follicles suggested a role in folliculogenesis. To study a presumed regulation of SNAP-25, we used a rat GC line (GFSHR-17), which expresses FSH receptors, and luteinizing human GC, which express LH receptors. FSH elevated SNAP-25 mRNA and protein levels about fivefold within 24 h in GFSHR-17 cells. The cAMP analogue dibutyryl-cAMP (db-cAMP) mimicked this action of FSH. The effects of both db-cAMP and FSH were inhibited by the protein kinase A (PKA) inhibitor H89. In contrast, SNAP-25 protein and mRNA-levels were not altered by LH/hCG in luteinized human GC. Our results for the first time identify SNAP-25b in oocytes and SNAP-25a in steroidogenic cells of the mammalian ovary. SNAP-25a and b may be involved in different exocytotic processes in these cell types.     

Hantavirus Gn and Gc Glycoproteins Self-Assemble into Virus-Like Particles

How hantaviruses assemble and exit infected cells remains largely unknown. Here, we show that the expression of Andes (ANDV) and Puumala (PUUV) hantavirus Gn and Gc envelope glycoproteins lead to their self-assembly into virus-like particles (VLPs) which were released to cell supernatants. The viral nucleoprotein was not required for particle formation. Further, a Gc endodomain deletion mutant did not abrogate VLP formation. The VLPs were pleomorphic, exposed protrusions and reacted with patient sera.     

The Two Na+ Sites in the Human Serotonin Transporter Play Distinct Roles in the Ion Coupling and Electrogenicity of Transport

Neurotransmitter transporters of the SLC6 family of proteins, including the human serotonin transporter (hSERT), utilize Na⁺, Cl⁻, and K⁺ gradients to induce conformational changes necessary for substrate translocation. Dysregulation of ion movement through monoamine transporters has been shown to impact neuronal firing potentials and could play a role in pathophysiologies, such as depression and anxiety. Despite multiple crystal structures of prokaryotic and eukaryotic SLC transporters indicating the location of both (or one) conserved Na⁺-binding sites (termed Na1 and Na2), much remains uncertain in regard to the movements and contributions of these cation-binding sites in the transport process. In this study, we utilize the unique properties of a mutation of hSERT at a single, highly conserved asparagine on TM1 (Asn-101) to provide several lines of evidence demonstrating mechanistically distinct roles for Na1 and Na2. Mutations at Asn-101 alter the cation dependence of the transporter, allowing Ca²⁺ (but not other cations) to functionally replace Na⁺ for driving transport and promoting 5-hydroxytryptamine (5-HT)-dependent conformational changes. Furthermore, in two-electrode voltage clamp studies in Xenopus oocytes, both Ca²⁺ and Na⁺ illicit 5-HT-induced currents in the Asn-101 mutants and reveal that, although Ca²⁺ promotes substrate-induced current, it does not appear to be the charge carrier during 5-HT transport. These findings, in addition to functional evaluation of Na1 and Na2 site mutants, reveal separate roles for Na1 and Na2 and provide insight into initiation of the translocation process as well as a mechanism whereby the reported SERT stoichiometry can be obtained despite the presence of two putative Na⁺-binding sites.     

Species effects on ecosystem processes are modified by faunal responses to habitat composition

Heterogeneity is a well-recognized feature of natural environments, and the spatial distribution and movement of individual species is primarily driven by resource requirements. In laboratory experiments designed to explore how different species drive ecosystem processes, such as nutrient release, habitat heterogeneity is often seen as something which must be rigorously controlled for. Most small experimental systems are therefore spatially homogeneous, and the link between environmental heterogeneity and its effects on the redistribution of individuals and species, and on ecosystem processes, has not been fully explored. In this paper, we used a mesocosm system to investigate the relationship between habitat composition, species movement and sediment nutrient release for each of four functionally contrasting species of marine benthic invertebrate macrofauna. For each species, various habitat configurations were generated by selectively enriching patches of sediment with macroalgae, a natural source of spatial variability in intertidal mudflats. We found that the direction and extent of faunal movement between patches differs with species identity, density and habitat composition. Combinations of these factors lead to concomitant changes in nutrient release, such that habitat composition effects are modified by species identity (in the case of NH4-N) and by species density (in the case of PO4-P). It is clear that failure to accommodate natural patterns of spatial heterogeneity in such studies may result in an incomplete understanding of system behaviour. This will be particularly important for future experiments designed to explore the effects of species richness on ecosystem processes, where the complex interactions reported here for single species may be compounded when species are brought together in multi-species combinations.     

The mechanistic basis for noncompetitive ibogaine inhibition of serotonin and dopamine transporters

Ibogaine, a hallucinogenic alkaloid proposed as a treatment for opiate withdrawal, has been shown to inhibit serotonin transporter (SERT) noncompetitively, in contrast to all other known inhibitors, which are competitive with substrate. Ibogaine binding to SERT increases accessibility in the permeation pathway connecting the substrate-binding site with the cytoplasm. Because of the structural similarity between ibogaine and serotonin, it had been suggested that ibogaine binds to the substrate site of SERT. The results presented here show that ibogaine binds to a distinct site, accessible from the cell exterior, to inhibit both serotonin transport and serotonin-induced ionic currents. Ibogaine noncompetitively inhibited transport by both SERT and the homologous dopamine transporter (DAT). Ibogaine blocked substrate-induced currents also in DAT and increased accessibility of the DAT cytoplasmic permeation pathway. When present on the cell exterior, ibogaine inhibited SERT substrate-induced currents, but not when it was introduced into the cytoplasm through the patch electrode. Similar to noncompetitive transport inhibition, the current block was not reversed by increasing substrate concentration. The kinetics of inhibitor binding and dissociation, as determined by their effect on SERT currents, indicated that ibogaine does not inhibit by forming a long-lived complex with SERT, but rather binds directly to the transporter in an inward-open conformation. A kinetic model for transport describing the noncompetitive action of ibogaine and the competitive action of cocaine accounts well for the results of the present study.     

Gap junction communication and connexin 43 gene expression in a rat granulosa cell line: regulation by follicle-stimulating hormone

Follicle-stimulating hormone is the major regulator of growth and development of antral follicles in the ovary. Granulosa cells (GCs) in these follicles are coupled via gap junctions (GJs) consisting of connexin 43 (Cx 43). Because we and others have found that Cx 43 and GJs, respectively, are more abundant in large antral follicles compared with small antral and preantral follicles, we hypothesized that FSH may control Cx 43 gene expression, GJ formation, and intercellular communication. To directly address these points, we chose a rat GC line (GFSHR-17) expressing the FSH receptor and the Cx 43 gene. The functionality of FSH receptors was shown by the effects of porcine FSH, namely cell rounding, reduced cellular proliferation, and stimulation of progesterone production of GFSHR-17 cells, which are effects that were detectable within hours. Treatment with FSH also statistically significantly increased Cx 43 mRNA levels, as shown after 6 to 9 h in Northern blots. These effects were antedated by altered GJ communication, which was observed within seconds. Using a single-cell/whole-cell patch clamp technique, we showed that FSH rapidly and reversibly enhanced electrical cell coupling of GFSHR-17 cells. Increased GJ communication was associated with statistically significantly decreased phosphorylation of Cx 43, which was observed within 10 min after FSH addition, during immunoprecipitation experiments. Our results demonstrate, to our knowledge for the first time, that the gonadotropin FSH acutely and directly stimulates intercellular communication of GFSHR-17 cells through existing GJs. Moreover, FSH also increases levels of Cx 43 mRNA. These changes are associated with reduced proliferation and enhanced differentiation of GFSHR-17 cells. In vivo factors in addition to FSH may be involved in the regulation of GJ/GJ communication between GCs in the follicle, but our results suggest that improved cell-to-cell coupling, enhanced Cx 43 gene expression, and possibly, formation of new GJs are direct consequences of FSH receptor activation and may antedate and/or initiate the pivotal effects of FSH on GCs.     

An insertion/deletion polymorphism at the D15S63 locus in the critical Prader-Willi syndrome region in 15q11-13

The probe YR9AB detects a two-allele insertion/ deletion polymorphism at the D15S63 locus.     

Predation increases multiple components of microbial diversity in activated sludge communities

Protozoan predators form an essential component of activated sludge communities that is tightly linked to wastewater treatment efficiency. Nonetheless, very little is known how protozoan predation is channelled via bacterial communities to affect ecosystem functioning. Therefore, we experimentally manipulated protozoan predation pressure in activated-sludge communities to determine its impacts on microbial diversity, composition and putative functionality. Different components of bacterial diversity such as taxa richness, evenness, genetic diversity and beta diversity all responded strongly and positively to high protozoan predation pressure. These responses were non-linear and levelled off at higher levels of predation pressure, supporting predictions of hump-shaped relationships between predation pressure and prey diversity. In contrast to predation intensity, the impact of predator diversity had both positive (taxa richness) and negative (evenness and phylogenetic distinctiveness) effects on bacterial diversity. Furthermore, predation shaped the structure of bacterial communities. Reduction in top-down control negatively affected the majority of taxa that are generally associated with increased treatment efficiency, compromising particularly the potential for nitrogen removal. Consequently, our findings highlight responses of bacterial diversity and community composition as two distinct mechanisms linking protozoan predation with ecosystem functioning in activated sludge communities.Subject terms: Microbial communities, Biodiversity     

Habitat structure mediates biodiversity effects on ecosystem properties

Much of what we know about the role of biodiversity in mediating ecosystem processes and function stems from manipulative experiments, which have largely been performed in isolated, homogeneous environments that do not incorporate habitat structure or allow natural community dynamics to develop. Here, we use a range of habitat configurations in a model marine benthic system to investigate the effects of species composition, resource heterogeneity and patch connectivity on ecosystem properties at both the patch (bioturbation intensity) and multi-patch (nutrient concentration) scale. We show that allowing fauna to move and preferentially select patches alters local species composition and density distributions, which has negative effects on ecosystem processes (bioturbation intensity) at the patch scale, but overall positive effects on ecosystem functioning (nutrient concentration) at the multi-patch scale. Our findings provide important evidence that community dynamics alter in response to localized resource heterogeneity and that these small-scale variations in habitat structure influence species contributions to ecosystem properties at larger scales. We conclude that habitat complexity forms an important buffer against disturbance and that contemporary estimates of the level of biodiversity required for maintaining future multi-functional systems may need to be revised.