N-formyl peptide receptors internalize but do not recycle in the absence of arrestins

Arrestins mediate phosphorylation-dependent desensitization, internalization, and initiation of signaling cascades for the majority of G protein-coupled receptors (GPCRs). Many GPCRs undergo agonist-mediated internalization through arrestin-dependent mechanisms, wherein arrestin serves as an adapter between the receptor and endocytic proteins. To understand the role of arrestins in N-formyl peptide receptor (FPR) trafficking, we stably expressed the FPR in a mouse embryonic fibroblast cell line (MEF) that lacked endogenous arrestin 2 and arrestin 3 (arrestin-deficient). We compared FPR internalization and recycling kinetics in these cells to congenic wild type MEF cell lines. Internalization of the FPR was not altered in the absence of arrestins. Since the FPR remains associated with arrestins following internalization, we investigated whether the rate of FPR recycling was altered in arrestin-deficient cells. While the FPR was able to recycle in the wild type cells, receptor recycling was largely absent in the arrestin double knockout cells. Reconstitution of the arrestin-deficient line with either arrestin 2 or arrestin 3 restored receptor recycling. Confocal fluorescence microscopy studies demonstrated that in arrestin-deficient cells the FPR may become trapped in the perinuclear recycling compartment. These observations indicate that, although the FPR can internalize in the absence of arrestins, recycling of internalized receptors to the cell surface is prevented. Our results suggest a novel role for arrestins in the post-endocytic trafficking of GPCRs.     

Monthly to interannual variability of microbial eukaryote assemblages at four depths in the eastern North Pacific

The monthly, seasonal and interannual variability of microbial eukaryote assemblages were examined at 5 m, the deep chlorophyll maximum, 150 m and 500 m at the San Pedro Ocean Time-series station (eastern North Pacific). The depths spanned transitions in temperature, light, nutrients and oxygen, and included a persistently hypoxic environment at 500 m. Terminal restriction fragment length polymorphism was used for the analysis of 237 samples that were collected between September 2000 and December 2010. Spatiotemporal variability patterns of microeukaryote assemblages indicated the presence of distinct shallow and deep communities at the SPOT station, presumably reflecting taxa that were specifically adapted for the conditions in those environments. Community similarity values between assemblages collected 1 month apart at each depth ranged between ∼20% and ∼84% (averages were ∼50–59%). The assemblage at 5 m was temporally more dynamic than deeper assemblages and also displayed substantial interannual variability during the first ∼3 years of the study. Evidence of seasonality was detected for the microbial eukaryote assemblage at 5 m between January 2008 and December 2010 and at 150 m between September 2000 and December 2003. Seasonality was not detected for assemblages at the deep chlorophyll a maximum, which varied in depth seasonally, or at 500 m. Microbial eukaryote assemblages exhibited cyclical patterns in at least 1 year at each depth, implying an annual resetting of communities. Substantial interannual variability was detected for assemblages at all depths and represented the largest source of temporal variability in this temperate coastal ecosystem.     

The phantom burster model for pancreatic beta-cells

Pancreatic beta-cells exhibit bursting oscillations with a wide range of periods. Whereas periods in isolated cells are generally either a few seconds or a few minutes, in intact islets of Langerhans they are intermediate (10-60 s). We develop a mathematical model for beta-cell electrical activity capable of generating this wide range of bursting oscillations. Unlike previous models, bursting is driven by the interaction of two slow processes, one with a relatively small time constant (1-5 s) and the other with a much larger time constant (1-2 min). Bursting on the intermediate time scale is generated without need for a slow process having an intermediate time constant, hence phantom bursting. The model suggests that isolated cells exhibiting a fast pattern may nonetheless possess slower processes that can be brought out by injecting suitable exogenous currents. Guided by this, we devise an experimental protocol using the dynamic clamp technique that reliably elicits islet-like, medium period oscillations from isolated cells. Finally, we show that strong electrical coupling between a fast burster and a slow burster can produce synchronized medium bursting, suggesting that islets may be composed of cells that are intrinsically either fast or slow, with few or none that are intrinsically medium.     

Pup Shoving by Adult Naked Mole-Rats

Adult naked mole‐rats (Heterocephalus glaber) characteristically perform an unusual behavior toward young: they shove small pups frequently and vigorously around the nest. We studied 15 litters in five captive colonies to quantify which adults shove pups, changes in shoving frequencies as pups develop, how external disturbances affect pup‐shoving frequencies, and behavior of juveniles that were not shoved as pups. In all litters and colonies the breeding female shoved pups significantly more often than any other individual. Breeding females also shoved adult colony mates, but at far lower rates than they shoved pups. Breeding males shoved pups about half as often as did breeding females. Together, the parents shoved pups ten times more often than did nonbreeders. Frequencies of pup shoving peaked when pups were 3–4 wk old, roughly coincident with weaning. When colonies were disturbed experimentally, frequencies of pup shoving increased dramatically, whereas rates at which nonbreeding adults were shoved decreased sharply. We separated four newly‐weaned litters and raised half the pups apart from their colony. When these litters were reunited 4–9 wk later, the unshoved (experimental) pups were the same size as the frequently shoved (control) pups, but the unshoved pups were significantly less likely to flee from a disturbance. Shoving of small pups encourages them to flee from danger, and also may enforce weaning.     

F9 embryonal carcinoma cells can differentiate into endoderm-like cells

The mouse teratocarcinoma cell line, F9, has been used in many laboratories as the epitome of the “nullipotent” embryonal carcinoma cell line. However, careful inspection of F9 cultures reveals the presence of small numbers of cells which possess several properties of endoderm, particularly parietal endoderm, and which can be shown to derive from the embryonal carcinoma component. Furthermore, tumors of F9 cells include isolated patches of endoderm-like cells surrounded by a thick secretion resembling Reichert's membrane. The proportion of endoderm-like cells in F9 cultures can be increased to varying degrees by causing the cells to form aggregates and/or maintaining them at high density for several days, although the endoderm-like cells produced in these ways contribute very little to the formation of subcutaneous tumors from the resultant mixed cultures. Differentiated cell types other than endoderm are rarely observed in F9 monolayer or aggregate cultures, even after several weeks. Cloning studies support the view that most, if not all, F9 cells can differentiate, albeit at very low incidence.     

Association of urinary 15-F2t-isoprostane level with oxygen desaturation and carotid intima–media thickness in nonobese sleep apnea patients

Obstructive sleep apnea (OSA) is characterized by recurrent apnea during sleep that may unbalance oxidative stress, increasing atherosclerosis. Among oxidative stress markers, 15-F_2t-isoprostane is considered one of the most sensitive and specific metabolites of lipid peroxidation. To explore the relationship between urinary 15-F_2t-isoprostane with sleep apnea severity and carotid modifications in nonobese OSA patients, 31 nonobese sleep apnea patients were studied, along with 10 lean subjects without OSA. Patients were assessed by polysomnography, blood pressure measurement, and ultrasonography to determine the carotid intima–media thickness (IMT). Urinary 15-F_2t-isoprostanes were measured by liquid chromatography–tandem mass spectrometry. Urinary 15-F_2t-isoprostane concentrations were increased in severe OSA patients compared to control subjects (20.2 ± 7.3 vs 12.3 ± 2.8 ng/mmol creatinine; P = 0.020). Mean carotid IMT was correlated with 15-F_2t-isoprostane (r = 0.532; P < 0.001) and with the apnea–hypopnea index (r = 0.345; P = 0.029). 15-F_2t-Isoprostane level was related to the night time spent at SaO₂ < 90% (r = 0.478; P = 0.002), the apnea–hypopnea index (r = 0.465; P = 0.003), and the mean nocturnal SaO₂ (r = ? 0.424; P = 0.007). These results showed a relationship between lipid peroxidation, carotid intima–media thickness, and intermittent hypoxia in nonobese OSA patients, thus reinforcing the hypothesis that oxidative stress could be involved in the early atherosclerotic process.     

Microclimate and Propagule Availability are Equally Important for Rehabilitation of Dryland N-Fixing Lichens

In some arid regions, rehabilitation of whole system N-fixation may be strongly facilitated by the recovery of populations of the lichen genus Collema . Identification of the limits to recovery of Collema in apparently suitable habitat should inform selection of rehabilitation techniques. We simultaneously tested the relative importance of three hypothetical limits to Collema recovery: active erosion, resource limitation, and propagule scarcity. We found that in our experimental system, active erosion had no effect on short-term establishment of Collema, whereas propagule addition did enhance recovery and microhabitat (a resource availability gradient) also exerted a strong influence. It is possible that attempts to improve N cycling via re-establishment of Collema might be best served by developing economical means of simulating moister, cooler microhabitats, e.g., sloping soil or creating partial shade, which would favor the establishment of naturally dispersed propagules, rather than introducing propagules.     

Molecular characterization and mapping of ALMT1, the aluminium-tolerance gene of bread wheat (Triticum aestivum L.).

The major aluminum (Al) tolerance gene in wheat ALMT1 confers. An Al-activated efflux of malate from root apices. We determined the genomic structure of the ALMT1 gene and found it consists of 6 exons interrupted by 5 introns. Sequencing a range of wheat genotypes identified 3 alleles for ALMT1, 1 of which was identical to the ALMT1 gene from an Aegilops tauschii accession. The ALMT1 gene was mapped to chromosome 4DL using 'Chinese Spring' deletion lines, and loss of ALMT1 coincided with the loss of both Al tolerance and Al-activated malate efflux. Aluminium tolerance in each of 5 different doubled-haploid populations was found to be conditioned by a single major gene. When ALMT1 was polymorphic between the parental lines, QTL and linkage analyses indicated that ALMT1 mapped to chromosome 4DL and cosegregated with Al tolerance. In 2 populations examined, Al tolerance also segregated with a greater capacity for Al-activated malate efflux. Aluminium tolerance was not associated with a particular coding allele for ALMT1, but was significantly correlated with the relative level of ALMT1 expression. These findings suggest that the Al tolerance in a diverse range of wheat genotypes is primarily conditioned by ALMT1.     

High-selenium yeast supplementation in free-living North American men: no effect on thyroid hormone metabolism or body composition.

In a prior study, we observed decreased serum 3,3',5-triiodothyronine (T(3)), increased serum thyrotropin and increased body weight in five men fed 297 microg/d of selenium (Se) in foods naturally high in Se while confined in a metabolic research unit. In an attempt to replicate and confirm those observations, we conducted a randomized study of high-Se yeast supplements (300 microg/d) or placebo yeast administered to 42 healthy free-living men for 48 weeks. Serum thyroxine, T(3) and thyrotropin did not change in supplemented or control subjects. Body weight increased in both groups during the 48-week treatment period and remained elevated for the 48-week follow-up period. Body fat increased by 1.2 kg in both groups. Energy intake and voluntary activity levels were not different between the groups and remained unchanged during the treatment period. Dietary intakes of Se, macronutrients and micronutrients were not different between groups and remained unchanged during the treatment period. These results suggest that our previous observation of a hypothyroidal response to high-Se foods was confounded by some aspect of the particular foods used, or were merely chance observations. Because of the high dose and long administration period, the present study suggests that the effects of Se supplements on thyroid hormone metabolism and energy metabolism in healthy North American men with adequate Se status do not represent a significant risk for unhealthy weight gain.