The ectodomain of the luteinizing hormone receptor interacts with exoloop 2 to constrain the transmembrane region: studies using chimeric human and fly receptors.

Lutropin (LH) and follitropin (FSH) receptors belong to a group of leucine-rich repeat-containing, G protein-coupled receptors (LGRs) found in vertebrates and flies. We fused the ectodomain of human LH or FSH receptors to the transmembrane region of fly LGR2. The chimeric human/fly receptors, unlike their wild type counterparts, exhibited ligand-independent constitutive activity. Because ectodomains likely interact with exoloops to constrain the receptors, individual exoloops of the chimeric receptor containing the ectodomain of the LH receptor and transmembrane region of fly LGR2 was replaced with LH receptor sequences. Chimeric receptors with the ectodomain and exoloop 2, but not exoloop 1 or 3, from LH receptors showed decreases in constitutive activity, but ligand treatment stimulated cAMP production. Furthermore, substitution of key resides in the hinge region of fly LGR2 with LH receptor sequences led to constitutive receptor activation; however, concomitant substitution of the homologous exoloop 2 of the LH receptor decreased G(s) coupling. These results suggest that the hinge region of the LH receptor interacts with exoloop 2 to constrain the receptor in an inactive conformation whereas ligand binding relieves this constraint, leading to G(s) activation.     

Spread and persistence of a rugulosin-producing endophyte in Picea glauca seedlings

We have studied Picea glauca (white spruce) endophyte colonization and its affect on the growth of Choristoneura fumiferana (spruce budworm). Here we examine the spread and persistence of a rugulosin-producing endophyte and rugulosin in needles from trees maintained in the nursery, as well as in trees planted in a test field site. Additionally, we report toxicity of rugulosin against three P. glauca needle herbivores: C. fumiferana, Lambdina fiscellaria (hemlock looper) and Zeiraphera canadensis (spruce budmoth). Reduction in body weight for both the C. fumiferana and L. fiscellaria were observed at 25 and 50 μm, respectively, and head capsules were reduced at 100 and 150 μm. Z. canadensis larvae did not perform as well in tests due to an Aspergillus fumigatus infection, but were shown to be lighter when tested with 100 and 150 μm compared with controls. The endophyte and its toxin were shown to spread throughout the nursery-grown seedlings. After 3.5 and 4.5 y post-inoculation (one and two years in the test site), the inoculated endophyte and its toxin had remained present with an average rugulosin concentration of 1 μg g⁻¹.     

Pneumococcal Capsular Polysaccharide Structure Predicts Serotype Prevalence

There are 91 known capsular serotypes of Streptococcus pneumoniae. The nasopharyngeal carriage prevalence of particular serotypes is relatively stable worldwide, but the host and bacterial factors that maintain these patterns are poorly understood. Given the possibility of serotype replacement following vaccination against seven clinically important serotypes, it is increasingly important to understand these factors. We hypothesized that the biochemical structure of the capsular polysaccharides could influence the degree of encapsulation of different serotypes, their susceptibility to killing by neutrophils, and ultimately their success during nasopharyngeal carriage. We sought to measure biological differences among capsular serotypes that may account for epidemiological patterns. Using an in vitro assay with both isogenic capsule-switch variants and clinical carriage isolates, we found an association between increased carriage prevalence and resistance to non-opsonic neutrophil-mediated killing, and serotypes that were resistant to neutrophil-mediated killing tended to be more heavily encapsulated, as determined by FITC-dextran exclusion. Next, we identified a link between polysaccharide structure and carriage prevalence. Significantly, non-vaccine serotypes that have become common in vaccinated populations tend to be those with fewer carbons per repeat unit and low energy expended per repeat unit, suggesting a novel biological principle to explain patterns of serotype replacement. More prevalent serotypes are more heavily encapsulated and more resistant to neutrophil-mediated killing, and these phenotypes are associated with the structure of the capsular polysaccharide, suggesting a direct relationship between polysaccharide biochemistry and the success of a serotype during nasopharyngeal carriage and potentially providing a method for predicting serotype replacement.     

Microglia in cerebral ischemia: molecular actions and interactions

The precise role of microglia in stroke and cerebral ischemia has been the subject of debate for a number of years. Microglia are capable of synthesizing numerous soluble and membrane-bound biomolecules, some known to be neuroprotective, some neurotoxic, whereas others have less definitive bioactivities. The molecular mechanisms through which microglia activate these molecules have thus become an important area of ischemia research. Here we provide a survey review that summarizes the key actions of microglial factors in cerebral ischemia including complement proteins, chemokines, pro-inflammatory cytokines, neurotrophic factors, hormones, and proteinases, as well several important messenger molecules that play a part in how these factors respond to extracellular signals during ischemic injuries. We also provide some new perspectives on how microglial intracellular signaling may contribute to the seemingly contradictory roles of several microglial effector molecules.     

53BP1 promotes ATM activity through direct interactions with the MRN complex

The Mre11/Rad50/Nbs1 (MRN) complex has a central function in facilitating activation of the ATM protein kinase at sites of DNA double‐strand breaks (DSBs). However, several other factors are also required in human cells for efficient signalling through MRN and ATM, including the tumour suppressor proteins p53‐binding protein 1 (53BP1) and BRCA1. In this study, we investigate the functions of these mediator proteins in ATM activation and find that the presence of 53BP1 and BRCA1 can amplify the effects of MRN when interactions between MRN and ATM are compromised. This effect is dependent on a direct interaction between MRN and the tandem breast cancer carboxy‐terminal (BRCT) repeats in 53BP1, and is accompanied by hyper‐phosphorylation of both Nbs1 and 53BP1. We also find that the BRCT domains of 53BP1 affect the overall structure of 53BP1 multimers and that this structure is important for promoting ATM phosphorylation of substrates as well as for the repair of DNA DSBs in mammalian cells.     

Biochemical analysis of ecto-nucleotide pyrophosphatase phosphodiesterase activity in brain membranes indicates involvement of NPP1 isoenzyme in extracellular hydrolysis of diadenosine polyphosphates in central nervous system

Synaptosomes and plasma membranes obtained from rat brain display ectoenzymatic hydrolytic activity responsible for hydrolysis of the neurotransmitter/neuroregulatory nucleotides diadenosine polyphosphates. Intact synaptosomes and plasma and synaptic membranes isolated by sucrose-gradient ultracentrifugation from several brain regions (hypothalamus, hippocampus, temporal cortex, frontal cortex striatum and cerebellum) degraded the fluorogenic substrates diethenoadenosine polyphosphates up to ethenoadenosine as by-product. Purified ectoenzyme cleaved substrates always releasing the mononucleotide moieties ethenoadenosine 5'-monophosphate and the corresponding ethenoadenosine (n-1) 5'-phosphate. Ectoenzymatic hydrolysis reached maximal activity at pH 9.0 (pH range 6.5-9.0) and was activated by Ca(2+) and Mg(2+) ions, with maximal effects around 2.0 mM cation. EDTA drastically reduced activity and Zn(2+) was required for enzyme reactivation. Hydrolysis of substrates followed hyperbolic kinetics with K(m) values in the 3-10 microM range. Diadenosine polyphosphates and heparin behaved as competitive inhibitors in the enzymatic hydrolysis of diethenoadenosine polyphosphates and AMP, ATP, alpha,beta-methyleneADP, ADPbetaS ATPgammaS, beta,gamma-methyleneATP, suramin and diethyl pyrocarbonate were also inhibitors. Ectoenzymatic activity shared the typical characteristics of members of the ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP) family and inhibition data suggest that NPP1 ectoenzyme is involved in the cleavage of extracellular diadenosine polyphosphates in brain. Synaptic membranes from cerebellum, hypothalamus and hippocampus presented the highest activities and no activity differences were observed between young and aged animals. However, plasma membranes showed a more homogeneous distribution of ectoenzymatic activity but a general increase was detected in aged animals. Enhancement of ectoenzymatic diadenosine polyphosphate cleaving activity found in plasma membranes from old animals could play a deleterious role in aged brain by limiting neuroprotective effects reported for extracellular diadenosine tetraphosphate.     

Increasing phylogenetic resolution at low taxonomic levels using massively parallel sequencing of chloroplast genomes

Background

Mycoheterotrophic plants are considered to associate very specifically with fungi. Mycoheterotrophic orchids are mostly associated with ectomycorrhizal fungi in temperate regions, or with saprobes or parasites in tropical regions. Although most mycoheterotrophic orchids occur in the tropics, few studies have been devoted to them, and the main conclusions about their specificity have hitherto been drawn from their association with ectomycorrhizal fungi in temperate regions.

Results

We investigated three Asiatic Neottieae species from ectomycorrhizal forests in Thailand. We found that all were associated with ectomycorrhizal fungi, such as Thelephoraceae, Russulaceae and Sebacinales. Based on ¹³C enrichment of their biomass, they probably received their organic carbon from these fungi, as do mycoheterotrophic Neottieae from temperate regions. Moreover, ¹³C enrichment suggested that some nearby green orchids received part of their carbon from fungi too. Nevertheless, two of the three orchids presented a unique feature for mycoheterotrophic plants: they were not specifically associated with a narrow clade of fungi. Some orchid individuals were even associated with up to nine different fungi.

Conclusion

Our results demonstrate that some green and mycoheterotrophic orchids in tropical regions can receive carbon from ectomycorrhizal fungi, and thus from trees. Our results reveal the absence of specificity in two mycoheterotrophic orchid-fungus associations in tropical regions, in contrast to most previous studies of mycoheterotrophic plants, which have been mainly focused on temperate orchids.     

Distribution of microbial biomass and potential for anaerobic respiration in Hanford Site 300 Area subsurface sediment

Subsurface sediments were recovered from a 52-m-deep borehole cored in the 300 Area of the Hanford Site in southeastern Washington State to assess the potential for biogeochemical transformation of radionuclide contaminants. Microbial analyses were made on 17 sediment samples traversing multiple geological units: the oxic coarse-grained Hanford formation (9 to 17.4 m), the oxic fine-grained upper Ringold formation (17.7 to 18.1 m), and the reduced Ringold formation (18.3 to 52 m). Microbial biomass (measured as phospholipid fatty acids) ranged from 7 to 974 pmols per g in discrete samples, with the highest numbers found in the Hanford formation. On average, strata below 17.4 m had 13-fold less biomass than those from shallower strata. The nosZ gene that encodes nitrous oxide reductase (measured by quantitative real-time PCR) had an abundance of 5 to 17 relative to that of total 16S rRNA genes below 18.3 m and <5 above 18.1 m. Most nosZ sequences were affiliated with Ochrobactrum anthropi (97 sequence similarity) or had a nearest neighbor of Achromobacter xylosoxidans (90 similarity). Passive multilevel sampling of groundwater geochemistry demonstrated a redox gradient in the 1.5-m region between the Hanford-Ringold formation contact and the Ringold oxic-anoxic interface. Within this zone, copies of the dsrA gene and Geobacteraceae had the highest relative abundance. The majority of dsrA genes detected near the interface were related to Desulfotomaculum spp. These analyses indicate that the region just below the contact between the Hanford and Ringold formations is a zone of active biogeochemical redox cycling.