Cellular trafficking and degradation of erythropoietin and novel erythropoiesis stimulating protein (NESP)

Erythropoietin (Epo) is essential for the production of mature red blood cells, and recombinant Epo is commonly used to treat anemia, but how Epo is degraded and cleared from the body is not understood. Glycosylation of Epo is required for its in vivo bioactivity, although not for in vitro receptor binding or stimulation of Epo-dependent cell lines; Epo glycosylation actually reduces the affinity of Epo for the Epo receptor (EpoR). Interestingly, a hyperglycosylated analog of Epo, called novel erythropoiesis-stimulating protein (NESP), has a lower affinity than Epo for the EpoR but has greater in vivo activity and a longer serum half-life than Epo. We hypothesize that a major mechanism for degradation of Epo in the body occurs in cells expressing the Epo receptor, through receptor-mediated endocytosis of Epo followed by degradation in lysosomes, and therefore investigated the trafficking and degradation of Epo and NESP by EpoR-expressing cells. We show that Epo and NESP are degraded only by cultured cells that express the EpoR, and their receptor binding, dissociation, and trafficking properties determine their rates of intracellular degradation. Epo binds surface EpoR faster than NESP (k(on) = 5.0 x 10(8) m(-1) min(-1) versus 1.1 x 10(8) m(-1) min(-1)) but dissociates slower (k(off) = 0.029 min(-1) versus 0.042 min(-1)). Surface-bound Epo and NESP are internalized at the same rate (k(in) = 0.06 min(-1)), and after internalization 60% of each ligand is resecreted intact and 40% degraded. Our kinetic model of Epo and NESP receptor binding, intracellular trafficking, and degradation explains why Epo is degraded faster than NESP at the cellular level.     

Deletion of exon I of SMAD7 in mice results in altered B cell responses

The members of the TGF-beta superfamily, i.e., TGF-beta isoforms, activins, and bone morphogenetic proteins, regulate growth, differentiation, and apoptosis, both during embryonic development and during postnatal life. Smad7 is induced by the TGF-beta superfamily members and negatively modulates their signaling, thus acting in a negative, autocrine feedback manner. In addition, Smad7 is induced by other stimuli. Thus, it can fine-tune and integrate TGF-beta signaling with other signaling pathways. To investigate the functional role(s) of Smad7 in vivo, we generated mice deficient in exon I of Smad7, leading to a partial loss of Smad7 function. Mutant animals are viable, but significantly smaller on the outbred CD-1 mouse strain background. Mutant B cells showed an overactive TGF-beta signaling measured as increase of phosphorylated Smad2-positive B cells compared with B cells from wild-type mice. In agreement with this expected increase in TGF-beta signaling, several changes in B cell responses were observed. Mutant B cells exhibited increased Ig class switch recombination to IgA, significantly enhanced spontaneous apoptosis in B cells, and a markedly reduced proliferative response to LPS stimulation. Interestingly, LPS treatment reverted the apoptotic phenotype in the mutant cells. Taken together, the observed phenotype highlights a prominent role for Smad7 in development and in regulating the immune system's response to TGF-beta.     

Lost in space? Searching for directions in the spatial modelling of individuals,populations and species ranges

The workshop ‘Spatial models in animal ecology, management and conservation’ held at Silwood Park (UK), 9–11 March 2010, aimed to synthesize recent progress in modelling the spatial dynamics of individuals, populations and species ranges and to provide directions for research. It brought together marine and terrestrial researchers working on spatial models at different levels of organization, using empirical as well as theory-driven approaches. Different approaches, temporal and spatial scales, and practical constraints predominate at different levels of organization and in different environments. However, there are theoretical concepts and specific methods that can fruitfully be transferred across levels and systems, including: habitat suitability characterization, movement rules, and ways of estimating uncertainty.     

Immune response to the West Nile virus in aged non-human primates

Background

Risk of encephalitis from West Nile virus (WNV) infection increases dramatically with age. Understanding the basis of this susceptibility requires development of suitable animal models. Here, we investigated the immune response to WNV in old non-human primates.

Methodology/Principal Findings

We investigated clinical, immunological and virological correlates of WNV infection in aging non-human primates. Aged (17–30yrs) and adult (6–9yrs) Rhesus macaques (RM) were challenged with WNV in the presence or the absence of the mosquito salivary gland extract (SGE) to approximate natural infection. None of the 26 animals exhibited clinical signs of the disease. Quantitative PCR suggested discrete and short-lived viremia, but infectious virus was never isolated. There was markedly increased, age-independent, proliferation of CD3⁻ non-B cells, followed by B-cell proliferation, which correlated to the loss of detectable WNV genomes. Moreover, animals primed with mosquito salivary gland extract exhibited reduced circulating WNV RNA. While we found the expected age-associated reduction in T cell proliferation, adaptive immunity did not correlate with infection outcome. That was further confirmed in a cohort of thymectomized and/or CD8 T-cell depleted Cynomolgus macaques (CM; N = 15), who also failed to develop WNV disease.

Conclusions/significance

Results are consistent with strong and age-independent innate resistance of macaques against WNV challenge. This animal model is therefore not suitable for vaccine and therapeutic testing against WNV. However, understanding the basis of their innate resistance against WNV in macaques could provide helpful clues to improve anti-WNV protection of older adults.     

1,2-diamino-ethane-substituted-6,7,8,9-tetrahydro-5H-pyrimido[4,5-d]azepines as TRPV1 antagonists with improved properties

Based upon a previously reported lead compound 1, a series of 1,2-diamino-ethane-substituted-6,7,8,9-tetrahydro-5H-pyrimido[4,5-d]azepines were synthesized and evaluated for improved physiochemical and pharmacokinetic properties while maintaining TRPV1 antagonist activity. Structure-activity relationship studies directed toward improving the aqueous solubility (pH 2 and fasted-state simulated intestinal fluid (SIF)) and rat pharmacokinetics led to the discovery of compound 13. Aqueous solubility of compound 13 (pH 2 ≥237 μg/mL and SIF=11 μg/mL) was significantly improved over compound 1 (pH 2=5 μg/mL and SIF=0.5 μg/mL). In addition, compound 13 afforded improved rat pharmacokinetics (CL=0.7 L/kg/h) compared to compound 1 (CL=3.1 L/kg/h). Compound 13 was orally bioavailable and afforded a significant reversal of carrageenan-induced thermal hyperalgesia at 5 and 30 mg/kg in rats.     

Seed mix design and first year management influence multifunctionality and cost‐effectiveness in prairie reconstruction

Agricultural intensification continues to diminish many ecosystem services in the North American Corn Belt. Conservation programs may be able to combat these losses more efficiently by developing initiatives that attempt to balance multiple ecological benefits. In this study, we examine how seed mix design and first year management influence three ecosystem services commonly provided by tallgrass prairie reconstructions (erosion control, weed resistance, and pollinator resources). We established research plots with three seed mixes, with and without first year mowing. The grass‐dominated “Economy” mix had 21 species and a 3:1 grass‐to‐forb seeding ratio. The forb‐dominated “Pollinator” mix had 38 species and a 1:3 grass‐to‐forb seeding ratio. The grass:forb balanced “Diversity” mix, which was designed to resemble regional prairie remnants, had 71 species and a 1:1 grass‐to‐forb ratio. To assess ecosystem services, we measured native stem density, cover, inflorescence production, and floral richness from 2015 to 2018. The Economy mix had high native cover and stem density, but produced few inflorescences and had low floral richness. The Pollinator mix had high inflorescence production and floral richness, but also had high bare ground and weed cover. The Diversity mix had high inflorescence production and floral richness (comparable to the Pollinator mix) and high native cover and stem density (comparable to the Economy mix). First year mowing accelerated native plant establishment and inflorescence production, enhancing the provisioning of ecosystem services during the early stages of a reconstruction. Our results indicate that prairie reconstructions with thoughtfully designed seed mixes can effectively address multiple conservation challenges.     

Contaminant loading in remote Arctic lakes affects cellular stress-related proteins expression in feral charr

The remote Arctic lakes on Bj?rn?ya Island, Norway, offer a unique opportunity to study possible affect of lifelong contaminant exposure in wild populations of landlocked Arctic charr (Salvelinus alpinus). This is because Lake Ellasj?en has persistent organic pollutant (POP) levels that are significantly greater than in the nearby Lake ?yangen. We examined whether this differential contaminant loading was reflected in the expression of protein markers of exposure and effect in the native fish. We assessed the expressions of cellular stress markers, including cytochrome P4501A (Cyp1A), heat shock protein 70 (hsp70), and glucocorticoid receptor (GR) in feral charr from the two lakes. The average polychlorinated biphenyl (PCB) load in the charr liver from Ellasj?en was approximately 25-fold higher than in individuals from ?yangen. Liver Cyp1A protein expression was significantly higher in individuals from Ellasj?en compared with ?yangen, confirming differential PCB exposure. There was no significant difference in hsp70 protein expression in charr liver between the two lakes. However, brain hsp70 protein expression was significantly elevated in charr from Ellasj?en compared with ?yangen. Also, liver GR protein expression was significantly higher in the Ellasj?en charr compared with ?yangen charr. Taken together, our results suggest changes to cellular stress-related protein expression as a possible adaptation to chronic-contaminant exposure in feral charr in the Norwegian high-Arctic.