Hypoxia regulates glutamate receptor trafficking through an HIF-independent mechanism

Oxygen influences behaviour in many organisms, with low levels (hypoxia) having devastating consequences for neuron survival. How neurons respond physiologically to counter the effects of hypoxia is not fully understood. Here, we show that hypoxia regulates the trafficking of the glutamate receptor GLR-1 in C. elegans neurons. Either hypoxia or mutations in egl-9, a prolyl hydroxylase cellular oxygen sensor, result in the internalization of GLR-1, the reduction of glutamate-activated currents, and the depression of GLR-1-mediated behaviours. Surprisingly, hypoxia-inducible factor (HIF)-1, the canonical substrate of EGL-9, is not required for this effect. Instead, EGL-9 interacts with the Mint orthologue LIN-10, a mediator of GLR-1 membrane recycling, to promote LIN-10 subcellular localization in an oxygen-dependent manner. The observed effects of hypoxia and egl-9 mutations require the activity of the proline-directed CDK-5 kinase and the CDK-5 phosphorylation sites on LIN-10, suggesting that EGL-9 and CDK-5 compete in an oxygen-dependent manner to regulate LIN-10 activity and thus GLR-1 trafficking. Our findings demonstrate a novel mechanism by which neurons sense and respond to hypoxia.     

Continental rain forest fragments in Singapore resist invasion by exotic plants

Abstract Aim In general, the plant communities of oceanic islands suffer more from exotic plant invasions than their continental equivalents. At least part of this difference may be contributed by differences in non‐biological factors, such as the antiquity and intensity of human impacts and the absence of internal barriers to dispersal, rather than differences in inherent invasibility. We tested the resistance of species‐rich continental rain forests to plant invasion on a small, continental island that has been subject to prolonged and intensive human impact. Location Singapore is a 683‐km² equatorial island <1 km from the Asian mainland and with a population of 4 million people. It has a continental biota but has been subject to human impacts as intense as on any oceanic island. Methods We sampled twenty‐nine sites in seven vegetation types, ranging from urban wasteland to fragments of primary lowland rain forest. In each sample plot, all plant species were identified, exotic cover was estimated, and a range of environmental variables measured. Additional qualitative surveys for exotic invasion were made in other forest areas in Singapore. The data were analysed by Spearman's rank correlation coefficient. Results The number of exotic species recorded at a site was unrelated to the number of native species. Across all sites, percentage canopy opening had the highest correlation with the number of exotic species, while soil pH (which largely reflects the incorporation of calcareous construction wastes) had the highest correlation if the mangrove sites were excluded. There were no exotics in mangrove forest and only a tropical American, bird‐dispersed shrub, Clidemia hirta (L.) D. Don (Melastomataceae: Koster's Curse), in primary and tall secondary forest patches. The species‐poor early stages of woody plant succession on highly degraded soils were also very resistant to exotic plant invasion. Main conclusions Long‐isolated rain forest fragments in an exotic‐dominated continental island landscape resist invasion by exotic plants, suggesting that the problems on oceanic islands may reflect an inherently greater invasibility. This study also adds to the increasing evidence that the floras of tropical rain forest fragments in South‐east Asia are remarkably resilient on a time‐scale of decades to a century or more.     

Isoenergetic penta- and hexanucleotide microarray probing and chemical mapping provide a secondary structure model for an RNA element orchestrating R2 retrotransposon protein function

LNA (locked nucleic acids, i.e. oligonucleotides with a methyl bridge between the 2′ oxygen and 4′ carbon of ribose) and 2,6-diaminopurine were incorporated into 2′-O-methyl RNA pentamer and hexamer probes to make a microarray that binds unpaired RNA approximately isoenergetically. That is, binding is roughly independent of target sequence if target is unfolded. The isoenergetic binding and short probe length simplify interpretation of binding to a structured RNA to provide insight into target RNA secondary structure. Microarray binding and chemical mapping were used to probe the secondary structure of a 323 nt segment of the 5′ coding region of the R2 retrotransposon from Bombyx mori (R2Bm 5′ RNA). This R2Bm 5′ RNA orchestrates functioning of the R2 protein responsible for cleaving the second strand of DNA during insertion of the R2 sequence into the genome. The experimental results were used as constraints in a free energy minimization algorithm to provide an initial model for the secondary structure of the R2Bm 5′ RNA.     

Effect of an acute ergotamine challenge on reproductive hormones in follicular phase heifers and progestin-treated cows

The objective of this research was to determine if ergotamine, an ergopeptine alkaloid isolated from Neotyphodium-infected grasses and associated with toxicoses in livestock, altered plasma concentrations of reproductive hormones in follicular phase heifers and in cows given a progestin implant. In Experiment 1, blood was sampled for 8h from four cycling heifers 2 days after synchronized luteolysis. Heifers were treated with ergotamine tartrate (19microg/kg) i.v. or saline vehicle in a simple cross-over design after 1h of pre-treatment blood sampling. Heifers received oxytocin (100USP units) i.v. 4h after ergotamine or saline treatment. Ergotamine reduced (P<0.01) prolactin concentrations from 1 to 4h post-treatment and increased (P<0.01) 13,14-dihydro-15-keto prostaglandin F2alpha (PGFM) concentrations from 2 to 5h post-treatment. A PGFM response to oxytocin was not detected. In Experiment 2, blood was sampled for 8h from six cycling cows 10 days after receiving a s.c. norgestomet implant. Cows were treated i.v. with ergotamine (20microg/kg) or saline in a simple cross-over design after 1h of pre-treatment blood sampling. Cows received gonadorelin (GnRH, 100microg) i.v. 1h after ergotamine or saline. Cows received oxytocin (100USP units) i.v. 4h after ergotamine or saline treatment. Ergotamine reduced (P<0.01) serum prolactin concentrations by 120min after treatment, with prolactin returning to pre-treatment concentrations by 200min after treatment. Saline-treated cows had lower (P<0.01) prolactin by 280min after treatment. Ergotamine-treated cows had higher (P<0.01) PGFM concentrations compared to saline-treated cows 120-240min after treatments, but the groups exhibited similar increases in PGFM after oxytocin. Plasma LH and FSH concentrations increased to peaks 100-120min after GnRH for both groups. However, the LH response to GnRH was greater (P<0.01) for ergotamine-treated cows. In summary, ergotamine lowered prolactin and elevated PGFM concentrations in follicular phase heifers and cows on norgestomet therapy. Ergotamine increased the LH response to exogenous GnRH in cows with norgestomet implants. These data highlight the potential of ergopeptine alkaloids to affect reproduction through altered endocrine function.     

Costs and response to conspecific brood parasitism by colonial red-breasted mergansers

Costs of conspecific brood parasitism (CBP) are expected to be influenced by a species’ life history traits. Precocial birds lay large clutches, and clutches that have been enlarged by CBP can affect host fitness through a longer incubation period, displaced eggs, and lower hatching success. We examined costs and response to CBP by hosts in a population of colonial red-breasted mergansers (Mergus serrator; n?=?400 nests over 8 years) within which 29% of parasitized clutches were enlarged considerably (≥?15 eggs). Length of the incubation period did not increase with clutch size. The mean number of eggs displaced from a parasitized nest during incubation (2.8) was 2×?greater than at an unparasitized nest (1.4). Hatching success declined by 2% for each additional egg in the nest. Thus, for a nest with?≥?15 eggs, one or more fewer host eggs hatch relative to an unparasitized nest with the same number of host eggs, assuming equal probability of success for all eggs. Hosts were 40% more likely to desert nests receiving 2 or 6 experimental eggs relative to unparasitized control nests, although it is unknown whether hens deserting a nest renested elsewhere. Our study indicates that costs of CBP to hosts during nesting may be limited to those red-breasted mergansers incubating the largest clutches (≥?15 eggs), and it raises questions about the adaptive significance of deserting a parasitized clutch.     

MLV integration site selection is driven by strong enhancers and active promoters

Retroviruses integrate into the host genome in patterns specific to each virus. Understanding the causes of these patterns can provide insight into viral integration mechanisms, pathology and genome evolution, and is critical to the development of safe gene therapy vectors. We generated murine leukemia virus integrations in human HepG2 and K562 cells and subjected them to second-generation sequencing, using a DNA barcoding technique that allowed us to quantify independent integration events. We characterized >3 700 000 unique integration events in two ENCODE-characterized cell lines. We find that integrations were most highly enriched in a subset of strong enhancers and active promoters. In both cell types, approximately half the integrations were found in <2% of the genome, demonstrating genomic influences even narrower than previously believed. The integration pattern of murine leukemia virus appears to be largely driven by regions that have high enrichment for multiple marks of active chromatin; the combination of histone marks present was sufficient to explain why some strong enhancers were more prone to integration than others. The approach we used is applicable to analyzing the integration pattern of any exogenous element and could be a valuable preclinical screen to evaluate the safety of gene therapy vectors.     

Adaptor Protein GRB2 Promotes Src Tyrosine Kinase Activation and Podosomal Organization by Protein-tyrosine Phosphatase ? in Osteoclasts

The non-receptor isoform of protein-tyrosine phosphatase ϵ (cyt-PTPe) supports adhesion of bone-resorbing osteoclasts by activating Src downstream of integrins. Loss of cyt-PTPe reduces Src activity in osteoclasts, reduces resorption of mineralized matrix both in vivo and in cell culture, and induces mild osteopetrosis in young female PTPe KO mice. Activation of Src by cyt-PTPe is dependent upon this phosphatase undergoing phosphorylation at its C-terminal Tyr-638 by partially active Src. To understand how cyt-PTPe activates Src, we screened 73 Src homology 2 (SH2) domains for binding to Tyr(P)-638 of cyt-PTPe. The SH2 domain of GRB2 bound Tyr(P)-638 of cyt-PTPe most prominently, whereas the Src SH2 domain did not bind at all, suggesting that GRB2 may link PTPe with downstream molecules. Further studies indicated that GRB2 is required for activation of Src by cyt-PTPe in osteoclast-like cells (OCLs) in culture. Overexpression of GRB2 in OCLs increased activating phosphorylation of Src at Tyr-416 and of cyt-PTPe at Tyr-638; opposite results were obtained when GRB2 expression was reduced by shRNA or by gene inactivation. Phosphorylation of cyt-PTPe at Tyr-683 and its association with GRB2 are integrin-driven processes in OCLs, and cyt-PTPe undergoes autodephosphorylation at Tyr-683, thus limiting Src activation by integrins. Reduced GRB2 expression also reduced the ability of bone marrow precursors to differentiate into OCLs and reduced the fraction of OCLs in which podosomal adhesion structures assume organization typical of active, resorbing cells. We conclude that GRB2 physically links cyt-PTPe with Src and enables cyt-PTPe to activate Src downstream of activated integrins in OCLs.