The structure and binding behavior of the bacterial cell surface layer protein SbsC

Surface layers (S-layers) comprise the outermost cell envelope component of most archaea and many bacteria. Here we present the structure of the bacterial S-layer protein SbsC from Geobacillus stearothermophilus, showing a very elongated and flexible molecule, with strong and specific binding to the secondary cell wall polymer (SCWP). The crystal structure of rSbsC((31-844)) revealed a novel fold, consisting of six separate domains, which are connected by short flexible linkers. The N-terminal domain exhibits positively charged residues regularly spaced along the putative ligand binding site matching the distance of the negative charges on the extended SCWP. Upon SCWP binding, a considerable stabilization of the N-terminal domain occurs. These findings provide insight into the processes of S-layer attachment to the underlying cell wall and self-assembly, and also accommodate the observed mechanical strength, the polarity of the S-layer, and the pronounced requirement for surface flexibility inherent to cell growth and division.     

Changes in subpopulations of boar sperm defined according to viability and plasma and acrosome membrane status observed during storage at 15 degrees C

Four boar ejaculates were preserved for 42 d at 15 °C to examine the changes produced in the quality of sperm membranes according to their response to a combined short hypoosmotic swelling test (sHOST) plus viability test designated the sHV test. Every 1 or 2 d, a sample from each ejaculate preserved in long-term extender was subjected to sperm motility determination and the sHV test. Through simultaneous examination by phase contrast and fluorescence microscopy, three subpopulations of sperm were identified according to their response to sHOST challenge and their viability status. In the subpopulations scoring positive in the sHOST, a further four sperm subpopulations were defined according to their viability and acrosome status. All the sperm subpopulations differed in terms of changes in their proportions produced during the course of preservation and individual differences among ejaculates were detected in terms of relationships shown among subpopulations. The combined sHOST/viability test was able to identify sperm subpopulations with the strongest plasma and acrosome membranes as well as a subpopulation of sperm that had undergone a true acrosome reaction.     

Identification of a Novel Function of PiT1 Critical for Cell Proliferation and Independent of Its Phosphate Transport Activity

PiT1 is a Na⁺-phosphate (P_i) cotransporter located at the plasma membrane that enables P_i entry into the cell. Its broad tissue expression pattern has led to the idea that together with the closely related family member PiT2, PiT1 is the ubiquitous supplier of P_i to the cell. Moreover, the role of P_i in phosphorylation reactions, ATP production, DNA structure, and synthesis has led to the view that P_i availability could be an important determinant of cell growth. However, these issues have not been clearly addressed to date, and the role of either P_i or PiT proteins in cell proliferation is unknown. Using RNA interference in HeLa and HepG2 cells, we show that transient or stable PiT1 depletion markedly reduces cell proliferation, delays cell cycle, and impairs mitosis and cytokinesis. In vivo, PiT1 depletion greatly reduced tumor growth when engineered HeLa cells were injected into nude mice. We provide evidence that this effect on cell proliferation is specific to PiT1 and not shared by PiT2 and is not the consequence of impaired membrane Na⁺-P_i transport. Moreover, we show that modulation of cell proliferation by PiT1 is independent from its transport function because the proliferation of PiT1-depleted cells can be rescued by non-transporting PiT1 mutants. PiT1 depletion leads to the phosphorylation of p38 mitogen-activated protein (MAP) kinase, whereas other MAP kinases and downstream targets of mammalian target of rapamycin (mTOR) remain unaffected. This study is the first to describe the effects of a P_i transporter in cell proliferation, tumor growth, and cell signaling.     

Spatial Distribution of Helicobacter spp. in the Gastrointestinal Tract of Dogs

Background:  In dogs, the gastric Helicobacter spp. have been well studied, but there is little information regarding the other parts of the alimentary system. We sought to determine the spatial distribution of Helicobacter spp. in the gastrointestinal tract and the hepatobiliary system of dogs using culture-independent methods.
Materials and methods:  Samples of stomach, duodenum, ileum, cecum, colon, pancreas, liver, and bile from six dogs were evaluated for Helicobacter spp. by genus, gastric, and enterohepatic Helicobacter spp. Polymerase chain reaction, 16S rRNA gene sequence analysis, immunohistochemistry, and fluorescence in situ hybridization (FISH).
Results:  In the stomach, Helicobacter spp. DNA was detected in all six dogs, with H. bizzozeronii and H. felis identified by specific polymerase chain reaction. Helicobacter organisms were localized within the surface mucus, the lumen of gastric glands, and inside parietal cells. The small intestine harbored gastric and enterohepatic Helicobacter spp. DNA/antigen in low amounts. In the cecum and colon, Helicobacter spp. DNA, with highest similarity to H. bilis /flexispira taxon 8, H. cinaedi , and H. canis, was detected in all six dogs. Helicobacter organisms were localized at the mucosal surface and within the crypts. Gastric Helicobacter spp. DNA was detected occasionally in the large intestine, but no gastric Helicobacter spp. were present in clone libraries or detected by FISH.
Conclusions:  This study demonstrates that in addition to the stomach, the large intestine of dogs is also abundantly colonized by Helicobacter spp. Additional studies are necessary to investigate the association between enterohepatic Helicobacter spp. and presence of intestinal inflammatory or proliferative disorders in dogs.     

Sheep Grazing Decreases Organic Carbon and Nitrogen Pools in the Patagonian Steppe: Combination of Direct and Indirect Effects

We explored the net effects of grazing on soil C and N pools in a Patagonian shrub–grass steppe (temperate South America). Net effects result from the combination of direct impacts of grazing on biogeochemical characteristics of microsites with indirect effects on relative cover of vegetated and unvegetated microsites. Within five independent areas, we sampled surface soils in sites subjected to three grazing intensities: (1) ungrazed sites inside grazing exclosures, (2) moderately grazed sites adjacent to them, and (3) intensely grazed sites within the same paddock. Grazing significantly reduced soil C and N pools, although this pattern was clearest in intensely grazed sites. This net effect was due to the combination of a direct reduction of soil N content in bare soil patches, and indirect effects mediated by the increase of the cover of bare soil microsites, with lower C and N content than either grass or shrub microsites. This increase in bare soil cover was accompanied by a reduction in cover of preferred grass species and standing dead material. Finally, stable isotope signatures varied significantly among grazed and ungrazed sites, with δ¹⁵N and δ¹³C significantly depleted in intensely grazed sites, suggesting reduced mineralization with increased grazing intensity. In the Patagonian steppe, grazing appears to exert a negative effect on soil C and N cycles; sound management practices must incorporate the importance of species shifts within life form, and the critical role of standing dead material in maintaining soil C and N stocks and biogeochemical processes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Author Contributions  RAG designed study, performed research, analyzed data, wrote the paper; ATA designed study, wrote the paper; CGGM designed study, performed research, analyzed data; MGP performed research; OES designed study; RBJ designed study, contributed new methods.     

Integration of tomato reproductive developmental landmarks and expression profiles, and the effect of SUN on fruit shape

Background  

Universally accepted landmark stages are necessary to highlight key events in plant reproductive development and to facilitate comparisons among species. Domestication and selection of tomato resulted in many varieties that differ in fruit shape and size. This diversity is useful to unravel underlying molecular and developmental mechanisms that control organ morphology and patterning. The tomato fruit shape gene SUN controls fruit elongation. The most dramatic effect of SUN on fruit shape occurs after pollination and fertilization although a detailed investigation into the timing of the fruit shape change as well as gene expression profiles during critical developmental stages has not been conducted.     

Regulated RalBP1 Binding to RalA and PSD-95 Controls AMPA Receptor Endocytosis and LTD

Long-term depression (LTD) is a long-lasting activity-dependent decrease in synaptic strength. NMDA receptor (NMDAR)–dependent LTD, an extensively studied form of LTD, involves the endocytosis of AMPA receptors (AMPARs) via protein dephosphorylation, but the underlying mechanism has remained unclear. We show here that a regulated interaction of the endocytic adaptor RalBP1 with two synaptic proteins, the small GTPase RalA and the postsynaptic scaffolding protein PSD-95, controls NMDAR-dependent AMPAR endocytosis during LTD. NMDAR activation stimulates RalA, which binds and translocates widespread RalBP1 to synapses. In addition, NMDAR activation dephosphorylates RalBP1, promoting the interaction of RalBP1 with PSD-95. These two regulated interactions are required for NMDAR-dependent AMPAR endocytosis and LTD and are sufficient to induce AMPAR endocytosis in the absence of NMDAR activation. RalA in the basal state, however, maintains surface AMPARs. We propose that NMDAR activation brings RalBP1 close to PSD-95 to promote the interaction of RalBP1-associated endocytic proteins with PSD-95-associated AMPARs. This suggests that scaffolding proteins at specialized cellular junctions can switch their function from maintenance to endocytosis of interacting membrane proteins in a regulated manner.     

Wild grapevine: silvestris,hybrids or cultivars that escaped from vineyards? Molecular evidence in Sardinia

Vitis vinifera ssp. silvestris, the spontaneous subspecies of V. vinifera L., is believed to be the ancestor of present grapevine cultivars. In this work, polymorphism at 13 SSR loci was investigated to answer the following key question: are wild plants (i) true silvestris, (ii) hybrids between wild and cultivated plants or (iii) or ‘escapes’ from vineyards? In particular, the objective of the present study was to identify truly wild individuals and to search for possible hybridization events. The study was performed in Sardinia, the second largest island in the Mediterranean Sea, which is characterized by a large and well‐described number of both grape cultivars and wild populations. This region was ideal for the study because of its spatial isolation and, consequently, limited contamination from outside material. The results of this study show that domesticated and wild grapevine germplasms are genetically divergent and thus are real silvestris. Pure lineages (both domesticated and wild) show very high average posterior probabilities of assignment to their own clusters, with a low level of introgression.