Modulation of the GABAA receptor by progesterone metabolites

The naturally occurring progesterone metabolites 5 beta-pregnan-3 alpha-ol-20-one and 5 beta-pregnane-3,20-dione reversibly enhance membrane currents elicited by locally applied GABA in bovine adrenomedullary chromaffin cells. Such potentiation was not influenced by the benzodiazepine antagonist Ro 15-1788. At concentrations in excess of those necessary to evoke potentiation of GABA currents, 5 beta-pregnan-3 alpha-ol-20-one and 5 beta-pregane-3,20-dione directly activated a membrane conductance. The resulting currents were potentiated by phenobarbitone and diazepam, and abolished by the GABAA-receptor antagonist, bicuculline. On outside-out membrane patches, 5 beta-pregnan-3 alpha-ol-20-one and 5 beta-pregnane-3,20-dione activated single channel currents of similar amplitude to those evoked by GABA. The results suggest that certain naturally occurring steroids potentiate the actions of GABA and, additionally, directly activate the GABAA receptor.     

Experts discuss cost-effective treatment of hypertension during world conference in Canada.

Even though an estimated 3.5 million Canadians are affected by hypertension, speakers at a World Conference on Hypertension Control questioned whether it is generally cost effective to treat younger men and women who have mild hypertension. Nonpharmacologic treatment via weight loss and lifestyle modification should be the first-line treatment, speakers stated. They looked at the basic principles for evaluating the economics of hypertension management and made recommendations on the cost effectiveness of treating various patient groups according to age and severity of their hypertension, and on the selection of diagnostic-evaluation procedures and antihypertensive medications.     

Stability and attractivity in associative memory networks

We focus on stable and attractive states in a network having two-state neuron-like elements. We calculate the connection matrix which guarantees the stability and the strongest attractivity of p memorized patterns. We present an analytical evaluation of the patterns' attractivity. These results are illustrated by some computer simulations.     

Animal board invited review: Factors affecting the early growth and development of gilt progeny compared to sow progeny

Progeny born to primiparous sows farrowing their first litter, often called gilt progeny (GP), are typically characterised by their poorer overall production performance than progeny from multiparous sows (sow progeny; SP). Gilt progeny consistently grow slower, are born and weaned lighter, and have higher postweaning illness and mortality rates than SP. Collectively, their poorer performance culminates in a long time to reach market weight and, ultimately, reduced revenue. Due to the high replacement rates of sows, the primiparous sow and her progeny represent a large proportion of the herd resulting in a significant loss for the pig industry. While the reasons for poorer performance are complex and multifaceted, they may largely be attributed to the immature age at which gilts are often mated and the significant impact of this on their metabolism during gestation and lactation. As a result, this can have negative consequences on the piglet itself. To improve GP performance, it is crucial to understand the biological basis for differences between GP and SP. The purpose of this review is to summarise published literature investigating differences in growth performance and health status between GP and SP. It also examines the primiparous sow during gestation and lactation and how the young sow must support her own growth while supporting the metabolic demands of her pregnancy and the growth and development of her litter. Finally, the underlying physiology of GP is discussed in terms of growth and development in utero, the neonatal period, and the early development of the gastrointestinal tract. The present review concludes that there are a number of interplaying factors relating to the anatomy and physiology of the primiparous sow and of GP themselves. The studies presented herein strongly suggest that poor support of piglet growth in utero and reduced colostrum and milk production and consumption are largely responsible for the underperformance of GP. It is therefore recommended that future management strategies focus on supporting the primiparous sow during gestation and lactation, increasing the preweaning growth of GP to improve their ability to cope with the stressors of weaning, selection of reproductive traits such as uterine capacity to improve birth weights and ultimately GP performance, and finally, increase the longevity of sows to reduce the proportion of GP entering the herd.     

Do Plant Cells Secrete Exosomes Derived from Multivesicular Bodies?

Multivesicular bodies (MVBs) are spherical endosomal organelles containing small vesicles formed by inward budding of the limiting membrane into the endosomal lumen. In mammalian red cells and cells of immune system, MVBs fuse with the plasma membrane in an exocytic manner, leading to release their contents including internal vesicles into the extracellular space. These released vesicles are termed exosomes. Transmission electron microscopy studies have shown that paramural vesicles situated between the plasma membrane and the cell wall occur in various cell wall-associated processes and are similar to exosomes both in location and in morphology. Our recent studies have revealed that MVBs and paramural vesicles proliferate when cell wall appositions are rapidly deposited beneath fungal penetration attempts or during plugging of plasmodesmata between hypersensitive cells and their intact neighboring cells. This indicates a potential secretion of exosome-like vesicles into the extracellular space by fusion of MVBs with the plasma membrane. This MVB-mediated secretion pathway was proposed on the basis of pioneer studies of MVBs and paramural vesicles in plants some forty years ago. Here, we recall the attention to the occurrence of MVB-mediated secretion of exosomes in plants.Key Words: cell wall, endocytosis, endosome, exocytosis, exosome, multivesicular body, paramural bodyMultivesicular bodies (MVBs) are spherical endosomal organelles containing a number of small vesicles formed by inward budding of the limiting membrane into the endosomal lumen.¹ MVBs contain endocytosed cargoes and deliver them into lysosomal/vacuolar compartments for degradation. They also incorporate newly synthesized proteins destined for lysosomal/vacuolar compartments.² In mammalian cells of hematopoietic origin, endosomal MVBs function in removal of endocytosed surface proteins in an exocytic manner. They are redirected to the plasma membrane, where they release their contents including internal vesicles into the extracellular space by membrane fusion. The released vesicles are termed exosomes.³ During reticulocyte maturation to erythrocyte, a group of surface proteins, such as the transferrin receptor, become obsolete and are discarded via MVB-mediated secretion.³ Time-course transmission electron microscopy (TEM) first revealed that colloidal gold-transferrin was internalized into MVBs via receptor-mediated endocytosis and then transferrin together with its receptor were delivered into the extracellular space via the fusion of MVBs with the plasma membrane of reticulocytes.⁴ Some other cell types of hematopoietic origin, such as activated platelets, cytotoxic T cells and antigen-presenting cells, also secrete exosomes. Exosomes thus may play a role in various physiological processes other than discarding obsolete proteins.³Our recent TEM studies provided ultrastructural evidence on the enhanced vesicle trafficking in barley leaf cells attacked by the biotrophic powdery mildew fungus. Multivesicular compartments including MVBs, intravacuolar MVBs, and paramural bodies turned out to proliferate in intact host cells during formation of cell wall appositions (papilla response), in the hypersensitive response, and during accommodation of haustoria.^5,6 MVBs proliferated in the cytoplasm of haustorium-containing epidermal cells during compatible interactions and near sites of cell wall-associated oxidative microburst either during the papilla response or during the hypersensitive response. Because MVBs in plant cells have been demonstrated to be endosomal compartments,^7–9 they may participate in internalization of nutrients from the apoplast of intact haustorium-containing epidermal cells and sequestration of damaged membranes and deleterious materials originating from the oxidative microburst.^5,6 The presence of intravacuolar MVBs with double limiting membranes (Fig. 1A) indicates an engulfment of MVBs by the tonoplast and a vacuole-mediated autophagy of MVBs.^5,6 MVBs, as prevacuolar compartments in plant cells,⁹ thus probably deliver their contents into the central vacuole via both the fusion with the tonoplast and the engulfment by the tonoplast (Fig. 2A and B). On the other hand, paramural bodies, in which small vesicles are situated between the cell wall and the plasma membrane, were associated with cell wall appositions deposited beneath fungal penetration attempts (Fig. 1B) or around hypersensitive cells including sites of plugged plasmodesmata (Fig. 1C and D).^5,6 Because paramural vesicles are similar to exosomes both in location and in morphology, we speculated that MVBs fuse with the plasma membrane in an exocytic manner to form paramural bodies.^5,6 Endocytosed cell surface materials in endosomal MVBs may be reused and delivered together with newly synthesized materials in Golgi apparatus-derived vesicles to cell wall appositions, which are deposited rapidly to prevent fungal penetration (Fig. 2A) or to contain hypersensitive cell death (Fig. 2B). MVBs thus may be driven along two distinct pathways to deliver their contents into either central vacuole or extracellular space.Open in a separate windowFigure 1Multivesicular compartments in intact cells in barley leaves attacked by the barley powdery mildew fungus. (A) An intravacuolar multivesicular body (MVB) with double limiting membranes in an intact epidermal cell (EC) adjacent to a hypersensitive epidermal cell (EC*). The arrows point to the outer limiting membrane, which is seemingly derived from the tonoplast. Note that neighboring intravacuolar vesicles (in between two arrowheads) may result from degradation of double limiting membranes of intravacuolar MVBs or may be delivered into the vacuole by MVB-fusion with the tonoplast. (B) Paramural vesicles (arrowheads) in a paramural body associated with cell wall appositions (asterisk) deposited by an intact epidermal cell. (C) A multivesicular body (MVB) in contact with a paramural body (PMB) (a nonmedian section) associated with cell wall appositions (asterisk) deposited by an intact mesophyll cell adjacent to a hypersensitive mesophyll cell. Note that cell wall appositions deposit beside an intercellular space (IS). The arrows point to the tonoplast. (D) A paramural body (PMB) associated with cell wall appositions (asterisks) blocking plasmodesmata (in between two arrowheads) at the side of an intact mesophyll cell (MC) underlying a hypersensitive epidermal cell (EC*). The arrows point to the tonoplast. CV, central vacuole; CW, cell wall; MB, microbody. Bars, 1µm.Open in a separate windowFigure 2Hypothetical diagram of delivery of endocytosed cell surface materials via MVBs into the central vacuole or the extracellular space where intact barley cells deposit cell wall appositions. (A) Deposition of cell wall appositions (asterisk) beneath powdery mildew penetration attempts. AGT, appressorial germ tube; PP, penetration peg. (B) Deposition of cell wall appositions (asterisks) against constricted plasmodesmata (PD) between a hypersensitive epidermal cell (EC) penetrated by the powdery mildew fungus and an underlying mesophyll cell (MC). H, haustorium. Arrows and numbers show pathways of vesicle trafficking. 1, Secretion of Golgi-derived vesicles containing newly synthesized materials; G, Golgi body; TGN, trans-Golgi network; 2, Endocytosis of cell surface materials from coated pits (coated open circles) via coated vesicles (coated circles) to multivesicular bodies (MVB); 3, Delivery of endocytosed materials for degradation inside the central vacuole (CV) via membrane fusion between MVBs and the tonoplast (T); small broken circles, vesicles in degradation; 4, Delivery of endocytosed materials for degradation inside the central vacuole via engulfment of MVBs by the tonoplast; large broken circles; MVB limiting membranes in degradation; 5, delivery of endocytosed materials into the extracellular space for deposition of cell wall appositions (asterisks) via membrane fusion between MVBs and the plasma membrane (PM). CW, cell wall; PMB, paramural body. PD0, 1, 2, 3 and 4 represent stages of plugging plasmodesmata. PD0, open plasmodesmata between two intact mesophyll cells (MC) subjacent to the hypersensitive epidermal cell (EC); PD1, constriction of plasmodesmata by callose (grey dots) deposition at plasmodesmal neck region; PD2, constricted plasmodesmata associated with plasmodesma-targeted secretion; PD3, further blocking of plasmodesmata by deposition of cell wall appositions; PD4, completely blocked plasmodesmata.Earlier than the discovery in animal cell systems,⁴ it was proposed in two independent papers in 1967 that the fusion of MVBs with the plasma membrane might result in the release of small vesicles into the extracellular space in fungi and in higher plants.^10,11 Several lines of evidence support the occurrence of MVB-mediated secretion of exosome-like vesicles in plants. First, vesicles of the same morphology as MVB internal vesicles have been observed in extracellular spaces or paramural spaces in various types of plant cells in various plant species by TEM.¹² An early study on endocytosis by soybean protoplasts also showed small extracellular vesicles attaching on the plasma membrane.⁸ Second, cooccurrence of MVBs and paramural vesicles has been observed in processes of cell proliferation, cell differentiation, and cell response to abiotic and biotic stress. Examples are cell plate formation,^13,14 secondary wall thickening,^15,16 cold hardness,^17,18 and deposition of cell wall appositions upon pathogen attack.^5,6,19–21 Third, identical molecular components, such as arabinogalactan proteins^22,23 and peroxidases,⁶ have been immunolocalized in both MVBs and paramural bodies. Despite these pieces of evidence, a conclusive demonstration of MVB-mediated secretion of exosomes in plants requires further exploration.The presently available experimental systems, approaches, and membrane markers may allow future demonstration of MVB-mediated secretion of exosomes in plants. Recent in vivo real-time observation and colocalization of cell surface and endosomal markers have already revealed that endosomes filled with endocytosed preexisting cell wall and plasma membrane materials are rapidly delivered to cytokinetic spaces to form cell plates in dividing tobacco, Arabidopsis, and maize cells.²⁴ Because TEM observed paramural bodies attaching to cell plates¹³ and MVBs in the vicinity of cell plates during all stages of cell plate formation,^14,25,26 MVBs and paramural bodies may participate in delivery of endocytosed building blocks to cell plates. Jiang''s and Robinson''s labs together developed a transgenic tobacco BY-2 cell line stably expressing a YFP-labeled vacuolar sorting receptor protein and antibodies against the vacuolar sorting receptor protein localized to the limiting membrane of MVBs.⁹ These tools together with live cell imaging and immunoelectron microscopy may allow visualization of MVB-fusion to the new plasma membrane, of vacuolar sorting receptors in both the limiting membrane of MVBs and the new plasma membrane, and of identical cell plate components in both internal vesicles of MVBs and paramural vesicles.In spite of obvious differences in plant and animal cytokinesis, the generation of cell plates by cell-plate-directed fusion of endosomes resembles the plugging of midbody canals by midbody-directed endosomes to separate daughter cells at the terminal phase of animal cytokinesis.²⁷ Likely, functional similarities of the fusion between endosomal MVBs and the plasma membrane to eliminate unwanted cell contents may also exist in maturation of mammalian red blood cells and plant sieve elements in the sense that the fusion of MVBs with the plasma membrane may occur during maturation of the latter.²⁸ On the other hand, although plant cells may secrete MVB-derived exosomes in defense response upon pathogen attack,^5,6 plant cell walls rule out the direct intercellular communication during the immune response mediated by exosomes in the circulation of mammals.³ In contrast, plasmodesma-directed secretion of exosomes would block the cell-to-cell communication between hypersensitive cells and their neighboring cells during hypersensitive response.⁵ Further exploration will lead us to a better understanding of similarities and differences of exosome secretion between plants and animals.     

Frasnian vertebrate taphonomy and sedimentology of macrofossil concentrations from the Langsēde Cliff,Latvia

Luk?evi?s, E., Ahlberg, P.E., Stinkulis, ?., Vasi?kova, J. & Zupi??, I. 2011: Frasnian vertebrate taphonomy and sedimentology of macrofossil concentrations from the Langsēde Cliff, Latvia. Lethaia, Vol. 45, pp. 356–370. The siliciclastic sequence of the Upper Devonian of Kurzeme, Western Latvia, is renowned for abundant vertebrate fossils, including the stem tetrapods Obruchevichthys gracilis and Ventastega curonica. During the first detailed taphonomic study of the vertebrate assemblage from the Ogre Formation cropping out at the Langsēde Cliff, Imula River, abundant vertebrate remains have been examined and identified as belonging to one psammosteid, two acanthodian and three sarcopterygian genera; the placoderm Bothriolepis maxima dominates the assemblage. Besides fully disarticulated placoderm and psammosteid plates, separate sarcopterygian scales and teeth, and acanthodian spines, partly articulated specimens including complete distal segments of Bothriolepis pectoral fins, Bothriolepis head shields and sarcopterygian lower jaws have been found. The size distribution of the placoderm bones demonstrates that the individuals within the assemblage are of approximately uniform age. Distinct zones have been traced within the horizontal distribution of the bones. These linear zones are almost perpendicular to the dominant dip azimuth of the cross‐beds and ripple‐laminae and most probably correspond to the depressions between subaqueous dunes. Concavity ratio varies significantly within the excavation area. The degree of fragmentation of the bones and disarticulation of the skeletons suggest that the carcasses were reworked and slightly transported before burial. Sedimentological data suggest deposition in a shallow marine environment under the influence of rapid currents. The fossiliferous bed consists of a basal bone conglomerate covered by a cross‐stratified sandstone with mud drapes, which is in turn overlain by ripple laminated sandstone, indicating the bones were buried by the gradual infilling of a tidal channel. All the Middle–Upper Devonian vertebrate bone‐beds from Latvia are associated with sandy to clayey deposits and have been formed in a sea‐coastal zone during rapid sedimentation episodes, but differ in fossil abundance and degree of preservation. □Agnathans, Devonian, facies analysis, fish, fossil assemblage, palaeoenvironment.     

Abundance and single-cell activity of heterotrophic bacterial groups in the western Arctic Ocean in summer and winter

Environmental conditions in the western Arctic Ocean range from constant light and nutrient depletion in summer to complete darkness and sea ice cover in winter. This seasonal environmental variation is likely to have an effect on the use of dissolved organic matter (DOM) by heterotrophic bacteria in surface water. However, this effect is not well studied and we know little about the activity of specific bacterial clades in the surface oceans. The use of DOM by three bacterial subgroups in both winter and summer was examined by microautoradiography combined with fluorescence in situ hybridization. We found selective use of substrates by these groups, although the abundances of Ant4D3 (Antarctic Gammaproteobacteria), Polaribacter (Bacteroidetes), and SAR11 (Alphaproteobacteria) were not different between summer and winter in the Beaufort and Chukchi Seas. The number of cells taking up glucose within all three bacterial groups decreased significantly from summer to winter, while the percentage of cells using leucine did not show a clear pattern between seasons. The uptake of the amino acid mix increased substantially from summer to winter by the Ant4D3 group, although such a large increase in uptake was not seen for the other two groups. Use of glucose by bacteria, but not use of leucine or the amino acid mix, related strongly to inorganic nutrients, chlorophyll a, and other environmental factors. Our results suggest a switch in use of dissolved organic substrates from summer to winter and that the three phylogenetic subgroups examined fill different niches in DOM use in the two seasons.     

Ubiquitin-dependent down-regulation of the neurokinin-1 receptor

Transient stimulation with substance P (SP) induces endocytosis and recycling of the neurokinin-1 receptor (NK(1)R). The effects of sustained stimulation by high concentrations of SP on NK(1)R trafficking and Ca(2+) signaling, as may occur during chronic inflammation and pain, are unknown. Chronic exposure to SP (100 nm, 3 h) completely desensitized Ca(2+) signaling by wild-type NK(1)R (NK(1)Rwt). Resensitization occurred after 16 h, and cycloheximide prevented resensitization, implicating new receptor synthesis. Lysine ubiquitination of G-protein-coupled receptors is a signal for their trafficking and degradation. Lysine-deficient mutant receptors (NK(1)RDelta5K/R, C-terminal tail lysines; and NK(1)RDelta10K/R, all intracellular lysines) were expressed at the plasma membrane and were functional because they responded to SP by endocytosis and by mobilization of Ca(2+) ions. SP desensitized NK(1)Rwt, NK(1)RDelta5K/R, and NK(1)RDelta10K/R. However, NK(1)RDelta5K/R and NK(1)RDelta10K/R resensitized 4-8-fold faster than NK(1)Rwt by cycloheximide-independent mechanisms. NK(1)RDelta325 (a naturally occurring truncated variant) showed incomplete desensitization, followed by a marked sensitization of signaling. Upon labeling receptors in living cells using antibodies to extracellular epitopes, we observed that SP induced endocytosis of NK(1)Rwt, NK(1)RDelta5K/R, and NK(1)RDelta10K/R. After 4 h in SP-free medium, NK(1)RDelta5K/R and NK(1)RDelta10K/R recycled to the plasma membrane, whereas NK(1)Rwt remained internalized. SP induced ubiquitination of NK(1)Rwt and NK(1)RDelta5K/R as determined by immunoprecipitation under nondenaturing and denaturing conditions and detected with antibodies for mono- and polyubiquitin. NK(1)RDelta10K/R was not ubiquitinated. Whereas SP induced degradation of NK(1)Rwt, NK(1)RDelta5K/R and NK(1)RDelta10K/R showed approximately 50% diminished degradation. Thus, chronic stimulation with SP induces ubiquitination of the NK(1)R, which mediates its degradation and down-regulation.     

Spatial and temporal population genetic structure of four northeastern Pacific littorinid gastropods: the effect of mode of larval development on variation at one mitochondrial and two nuclear DNA markers

We investigated the effect of development mode on the spatial and temporal population genetic structure of four littorinid gastropod species. Snails were collected from the same three sites on the west coast of Vancouver Island, Canada in 1997 and again in 2007. DNA sequences were obtained for one mitochondrial gene, cytochrome b ( Cyt b ), and for up to two nuclear genes, heat shock cognate 70 ( HSC70 ) and aminopeptidase N intron ( APN54 ). We found that the mean level of genetic diversity and long-term effective population sizes ( N _e) were significantly greater for two species, Littorina scutulata and L. plena , that had a planktotrophic larval stage than for two species, Littorina sitkana and L. subrotundata , that laid benthic egg masses which hatched directly into crawl-away juveniles. Predictably, two poorly dispersing species, L. sitkana and L. subrotundata , showed significant spatial genetic structure at an 11- to 65-km geographical scale that was not observed in the two planktotrophic species. Conversely, the two planktotrophic species had more temporal genetic structure over a 10-year interval than did the two direct-developing species and showed highly significant temporal structure for spatially pooled samples. The greater temporal genetic variation of the two planktotrophic species may have been caused by their high fecundity, high larval dispersal, and low but spatially correlated early survivorship. The sweepstakes-like reproductive success of the planktotrophic species could allow a few related females to populate hundreds of kilometres of coastline and may explain their substantially larger temporal genetic variance but lower spatial genetic variance relative to the direct-developing species.