Isolation and purification of afferent lymph dendritic cells that drain the skin of cattle

Dendritic cells (DCs) are central to the induction of immune responses and are a pivotal control point that determines the outcome of infectious challenge. Cannulation of afferent lymphatic vessels allows the isolation of large numbers of lymph DCs. First, lymph nodes that are draining the skin are surgically removed (takes approximately 1 h). Over a period of 6-8 weeks, afferent lymphatic vessels re-anastomose with the efferent duct, forming larger 'pseudoafferent' lymphatic vessels that can be surgically cannulated. Surgical cannulation takes 2 h to perform; daily maintenance of the catheter requires 30 min. Isolation of lymph cells requires 1 h and an additional 60-180 min to enrich or purify the DCs. The lymph can be harvested for up to 1 month, with relatively constant cell numbers and subset distribution throughout this period. This technique, although technically demanding, facilitates studies of DCs and other cells that traffic in the lymph in both the steady state and following antigenic exposure.     

Plasma adipokines and body composition in response to modest dietary manipulations in the mouse

Objective: The relationship between adipokine levels and body composition has not been carefully examined. Most studies in humans are cross‐sectional, and the few studies in mice have been restricted to a comparison of control animals with markedly obese, insulin‐resistant mice. Our objective was to study changes in adipokine levels and body composition in response to modest dietary intervention. Research Methods and Procedures: Plasma resistin, adiponectin, and leptin levels were examined in mice fed ad libitum, a 75% restricted diet, or a diet supplemented with 10% sucrose. Body composition was determined by whole‐body DXA. Results: The percentage body fat was reduced in mice subjected to the restricted diet and increased in mice supplemented with 10% dextrose. Adipokine levels were not different in either of these groups compared with the control mice. A significant inverse correlation was observed between resistin levels and total body fat, whereas there was no significant correlation between body fat and adiponectin levels. Positive correlations were observed between leptin levels and percentage body fat, total body fat, and abdominal fat. Leptin levels correlated with plasma glucose, but multivariate analysis revealed that this correlation was the result of a strong positive correlation between leptin and insulin levels. There were no correlations between glycemia and resistin or glycemia and adiponectin levels, and no correlation was observed between any of the adipokine levels and bone mineral content or density. Discussion: These data suggest that in the mouse, modest dietary perturbations have little effect on resistin and adiponectin levels despite significant effects on glycemia, insulin levels, and bone parameters.     

The mammalian alphaD-globin gene lineage and a new model for the molecular evolution of alpha-globin gene clusters at the stem of the mammalian radiation

We have explored the evolution of the alpha-globin gene family by comparative sequence and phylogenetic analyses of mammalian alpha-globin genes. Our analyses reveal the existence of a new alpha-globin gene lineage in mammals that is related to the alpha(D)-globin genes of birds, squamates and turtles. The gene is located in the middle of the alpha-globin gene cluster of a marsupial, Sminthopsis macroura and of humans. It exists in a wide variety of additional mammals, including pigs, cows, cats, and dogs, but is a pseudogene in American marsupials. Evolutionary analyses suggest that the gene has generally evolved under purifying selection, indicative of a functional gene. The presence of mRNA products in humans, pigs, and cows also suggest that the gene is expressed and likely to be functional. The analyses support the hypothesis that the alpha(D)-globin gene lineage has an ancient evolutionary origin that predates the divergence of amniotes. The structural similarity of alpha-globin gene clusters of marsupials and humans suggest that an eight gene cluster (5'-zeta2-zeta1-alpha(D)-alpha3-alpha2-alpha1-theta-omega-3'), including seven alpha-like genes and one beta-like globin gene (omega-globin) existed in the common ancestor of all marsupial and eutherian mammals. This basic structure has remained relatively stable in marsupials and in the lineage leading to humans, although omega-globin has been lost from the alpha-globin gene cluster of humans.     

Identifying protein interactions with metal-modified DNA using microarray technology

Protein microarrays have been used extensively to identify protein–protein interactions; however, this technology has not been widely applied to protein–DNA interactions. In particular, this work demonstrates the utility of this technique for rapidly identifying interactions of proteins with metal-modified DNA. Protein macroarray experiments were carried out with high mobility group protein 1 (HMG-1) and cisplatin- and chromium-modified 50-mer oligonucleotides to demonstrate “proof of principle.” Commercially available protein microarrays containing many different classes of human proteins were then employed to search for additional interactions with cisplatin-modified DNA. The results of the microarray experiments confirmed some known interactions and, more importantly, identified many novel protein interactions, demonstrating the utility of this method as a rapid, high-throughput technique to discover proteins that interact with metal-modified DNA. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

LIFE HISTORY AND THE EVOLUTION OF PARENTAL CARE

Patterns of parental care are strikingly diverse in nature, and parental care is thought to have evolved repeatedly multiple times. Surprisingly, relatively little is known about the most general conditions that lead to the origin of parental care. Here, we use a theoretical approach to explore the basic life‐history conditions (i.e., stage‐specific mortality and maturation rates, reproductive rates) that are most likely to favor the evolution of some form of parental care from a state of no care. We focus on parental care of eggs and eggs and juveniles and consider varying magnitudes of the benefits of care. Our results suggest that parental care can evolve under a range of life‐history conditions, but in general will be most strongly favored when egg death rate in the absence of care is high, juvenile survival in the absence of care is low (for the scenario in which care extends into the juvenile stage), adult death rate is relatively high, egg maturation rate is low, and the duration of the juvenile stage is relatively short. Additionally, parental care has the potential to be favored at a broad range of adult reproductive rates. The relative importance of these life‐history conditions in favoring or limiting the evolution of care depends on the magnitude of the benefits of care, the relationship between initial egg allocation and subsequent offspring survival, and whether care extends into the juvenile stage. The results of our model provide a general set of predictions regarding when we would expect parental care to evolve from a state of no care, and in conjunction with other work on the topic, will enhance our understanding of the evolutionary dynamics of parental care and facilitate comparative analyses.     

Signs of mood and anxiety disorders in chimpanzees

Background

In humans, traumatic experiences are sometimes followed by psychiatric disorders. In chimpanzees, studies have demonstrated an association between traumatic events and the emergence of behavioral disturbances resembling posttraumatic stress disorder (PTSD) and depression. We addressed the following central question: Do chimpanzees develop posttraumatic symptoms, in the form of abnormal behaviors, which cluster into syndromes similar to those described in human mood and anxiety disorders?

Methodology/Principal Findings

In phase 1 of this study, we accessed case reports of chimpanzees who had been reportedly subjected to traumatic events, such as maternal separation, social isolation, experimentation, or similar experiences. We applied and tested DSM-IV criteria for PTSD and major depression to published case reports of 20 chimpanzees identified through PrimateLit. Additionally, using the DSM-IV criteria and ethograms as guides, we developed behaviorally anchored alternative criteria that were applied to the case reports. A small number of chimpanzees in the case studies met DSM-IV criteria for PTSD and depression. Measures of inter-rater reliability, including Fleiss'' kappa and percentage agreement, were higher with use of the alternative criteria for PTSD and depression. In phase 2, the alternative criteria were applied to chimpanzees living in wild sites in Africa (n = 196) and chimpanzees living in sanctuaries with prior histories of experimentation, orphanage, illegal seizure, or violent human conflict (n = 168). In phase 2, 58% of chimpanzees living in sanctuaries met the set of alternative criteria for depression, compared with 3% of chimpanzees in the wild (p = 0.04), and 44% of chimpanzees in sanctuaries met the set of alternative criteria for PTSD, compared with 0.5% of chimpanzees in the wild (p = 0.04).

Conclusions/Significance

Chimpanzees display behavioral clusters similar to PTSD and depression in their key diagnostic criteria, underscoring the importance of ethical considerations regarding the use of chimpanzees in experimentation and other captive settings.     

The domains required to direct core proteins of hepatitis C virus and GB virus-B to lipid droplets share common features with plant oleosin proteins.

In mammalian tissue culture cells, the core protein of hepatitis C virus (HCV) is located at the surface of lipid droplets, which are cytoplasmic structures that store lipid. The critical amino acid sequences necessary for this localization are in a region of core protein that is absent in flavi- and pestiviruses, which are related to HCV. From our sequence comparisons, this region in HCV core was present in the corresponding protein of GBV-B, another virus whose genomic sequence has significant similarity to HCV. Expression of the putative GBV-B core protein revealed that it also was directed to lipid droplets. By extending the comparisons to cellular proteins, there were amino acid sequence similarities between the domains for lipid droplet association in HCV core and plant oleosin proteins. To determine whether these similarities were related functionally, an oleosin encoded by the Brassica napus bniii gene was expressed in different mammalian cell lines, where it retained the capacity to bind to lipid droplets. Analysis of deletion mutants indicated that the critical region within the protein required for this localization was the same for both plant and mammalian cells. A common feature in the viral and plant sequences was a motif containing proline residues. Mutagenesis of these residues in HCV core and plant oleosin abolished lipid droplet association. Finally, the domain within HCV core required for binding to lipid droplets could substitute for the equivalent domain in oleosin, further indicating the functional relatedness between the viral and plant sequences. These studies identify common features in disparate proteins that are required for lipid droplet localization.     

N2-fixation by methanotrophs sustains carbon and nitrogen accumulation in pristine peatlands

Symbiotic relationships between N₂-fixing prokaryotes and their autotrophic hosts are essential in nitrogen (N)-limited ecosystems, yet the importance of this association in pristine boreal peatlands, which store 25 % of the world’s soil (C), has been overlooked. External inputs of N to bogs are predominantly atmospheric, and given that regions of boreal Canada anchor some of the lowest rates found globally (~1 kg N ha^?1 year^?1), biomass production is thought to be limited primarily by N. Despite historically low N deposition, we show that boreal bogs have accumulated approximately 12–25 times more N than can be explained by atmospheric inputs. Here we demonstrate high rates of biological N₂-fixation in prokaryotes associated with Sphagnum mosses that can fully account for the missing input of N needed to sustain high rates of C sequestration. Additionally, N amendment experiments in the field did not increase Sphagnum production, indicating that mosses are not limited by N. Lastly, by examining the composition and abundance of N₂-fixing prokaryotes by quantifying gene expression of 16S rRNA and nitrogenase-encoding nifH, we show that rates of N₂-fixation are driven by the substantial contribution from methanotrophs, and not from cyanobacteria. We conclude biological N₂-fixation drives high sequestration of C in pristine peatlands, and may play an important role in moderating fluxes of methane, one of the most important greenhouse gases produced in peatlands. Understanding the mechanistic controls on biological N₂-fixation is crucial for assessing the fate of peatland carbon stocks under scenarios of climate change and enhanced anthropogenic N deposition.     

Stressful environments can indirectly select for increased longevity

Longevity is modulated by a range of conserved genes in eukaryotes, but it is unclear how variation in these genes contributes to the evolution of longevity in nature. Mutations that increase life span in model organisms typically induce trade‐offs which lead to a net reduction in fitness, suggesting that such mutations are unlikely to become established in natural populations. However, the fitness consequences of manipulating longevity have rarely been assessed in heterogeneous environments, in which stressful conditions are encountered. Using laboratory selection experiments, we demonstrate that long‐lived, stress‐resistant Caenorhabditis elegans age‐1(hx546) mutants have higher fitness than the wild‐type genotype if mixed genotype populations are periodically exposed to high temperatures when food is not limited. We further establish, using stochastic population projection models, that the age‐1(hx546) mutant allele can confer a selective advantage if temperature stress is encountered when food availability also varies over time. Our results indicate that heterogeneity in environmental stress may lead to altered allele frequencies over ecological timescales and indirectly drive the evolution of longevity. This has important implications for understanding the evolution of life‐history strategies.