Transmembrane potential and ionic content of rat alveolar macrophages

The cell volume, cell water, intracellular ionic concentrations, and transmembrane potential of rat alveolar macrophages were determined. The measurements were made on cells which had been separated from the medium by centrifugation through dibutyl phthalate in order to greatly reduce the trapped extracellular space. The mean cell volume of the alveolar macrophages is 1,525 cubic microns and 72% of this volume is water. The intracellular fluid is high in Na+ (97 mM) and lower in K+ (50 mM) and the intracellular Cl- concentration in 64 mM. The transmembrane potential, as measured from the equilibrium distribution of tritiated triphenylmethyl phosphonium and by using the fluorescent probe, Di-S-C3(5), is approximately -37 millivolts. Neither Na+, K+, nor Cl- is distributed at equilibrium. However, the K+ permeability of alveolar macrophage membranes appears to be greater than Na+ permeability.     

Extranucleosomal DNA binding directs nucleosome sliding by Chd1

Chd1- and ISWI-type chromatin remodelers can sense extranucleosomal DNA and preferentially shift nucleosomes toward longer stretches of available DNA. The DNA-binding domains of these chromatin remodelers are believed to be responsible for sensing extranucleosomal DNA and are needed for robust sliding, but it is unclear how these domains contribute to directional movement of nucleosomes. Here, we show that the DNA-binding domain of Chd1 is not essential for nucleosome sliding but is critical for centering mononucleosomes on short DNA fragments. Remarkably, nucleosome centering was achieved by replacing the native DNA-binding domain of Chd1 with foreign DNA-binding domains of Escherichia coli AraC or Drosophila melanogaster engrailed. Introducing target DNA sequences recognized by the foreign domains enabled the remodelers to rapidly shift nucleosomes toward these binding sites, demonstrating that these foreign DNA-binding domains dictated the direction of sliding. Sequence-directed sliding occluded the target DNA sequences on the nucleosome enough to promote release of the remodeler. Target DNA sequences were highly stimulatory at multiple positions flanking the nucleosome and had the strongest influence when separated from the nucleosome by 23 or fewer base pairs. These results suggest that the DNA-binding domain's affinity for extranucleosomal DNA is the key determinant for the direction that Chd1 shifts the nucleosome.     

The human dimension of fire regimes on Earth

Humans and their ancestors are unique in being a fire-making species, but 'natural' (i.e. independent of humans) fires have an ancient, geological history on Earth. Natural fires have influenced biological evolution and global biogeochemical cycles, making fire integral to the functioning of some biomes. Globally, debate rages about the impact on ecosystems of prehistoric human-set fires, with views ranging from catastrophic to negligible. Understanding of the diversity of human fire regimes on Earth in the past, present and future remains rudimentary. It remains uncertain how humans have caused a departure from 'natural' background levels that vary with climate change. Available evidence shows that modern humans can increase or decrease background levels of natural fire activity by clearing forests, promoting grazing, dispersing plants, altering ignition patterns and actively suppressing fires, thereby causing substantial ecosystem changes and loss of biodiversity. Some of these contemporary fire regimes cause substantial economic disruptions owing to the destruction of infrastructure, degradation of ecosystem services, loss of life, and smoke-related health effects. These episodic disasters help frame negative public attitudes towards landscape fires, despite the need for burning to sustain some ecosystems. Greenhouse gas-induced warming and changes in the hydrological cycle may increase the occurrence of large, severe fires, with potentially significant feedbacks to the Earth system. Improved understanding of human fire regimes demands: (1) better data on past and current human influences on fire regimes to enable global comparative analyses, (2) a greater understanding of different cultural traditions of landscape burning and their positive and negative social, economic and ecological effects, and (3) more realistic representations of anthropogenic fire in global vegetation and climate change models. We provide an historical framework to promote understanding of the development and diversification of fire regimes, covering the pre-human period, human domestication of fire, and the subsequent transition from subsistence agriculture to industrial economies. All of these phases still occur on Earth, providing opportunities for comparative research.     

Hot Spots of Inorganic Nitrogen Availability in an Alpine-Subalpine Ecosystem,Colorado Front Range

Inorganic nitrogen (N) availability hot spots have been documented in many ecosystems, but major uncertainties remain about their prevalence, timing, and causes. Using a novel mathematical definition of hot spots, spatially explicit measurements of KCl-extractable inorganic N, 2-week soil incubations in the field, ion-exchange resins deployed for 1 year, and a set of associated biotic and abiotic variables, we investigated inorganic N availability hot spots within a 0.89 km² alpine-subalpine ecosystem in the Colorado Front Range. Measurements of KCl-extractable NH₄ ⁺ and NO₃ ⁻ taken on multiple dates showed that hot spots of N availability were present in some but not all parts of the study site and that hot spot location varied over the course of the season. Ion-exchange resins showed that over a 1-year period hot spots were important contributors to resin-available N at the landscape level, with 14% of resin locations accounting for 58% of total resin-extractable inorganic N. The KCl-extractable and resin-available inorganic N measurements showed that although spatial variation in the timing of hot spots (that is, hot moments) spreads the influence of short-term hot spots across the landscape to some extent, spatial variation in inorganic N availability is still important when integrated over 1 year. Resin-available N was poorly correlated with the biotic and abiotic variables that we measured, though we did observe that hot spots of resin-available N were twice as common below tree and shrub canopies than in herbaceous areas. Beyond this relationship with canopy structure, neither KCl-extractable nor resin-available inorganic N hot spots were closely related to plant species identity. Instead, the most effective predictor of KCl-extractable NH₄ ⁺ was the size of the soil organic matter (SOM) N pool, with nearly all hot spots appearing in soils that had greater than 1.4% SOM N.     

Heart rate and perceived exertion during self-selected intensities for exergaming compared to traditional exercise in college-age participants

Exergames may be useful for promoting physical activity in younger populations. Heart rate (HRs) responses and rating of perceived exertion (RPE) at self-selected intensities were compared in college-age participants during 2 modes of exergame activity vs. traditional exercise. Thirty-seven participants (men: 20, women: 17) completed 3 30-minute self-selected intensity trials: (a) video game interactive bicycle ergometer (GB) (CatEye GB300), (b) interactive video dance game (Dance Dance Revolution [DDR]), and (c) traditional cycle ergometer (CE) while watching television. Mean HR, peak HR (PkHR), and minutes above target HR (THR) were significantly higher for GB (144 ± 22 b · min(-1) [57% HR reserve (HRR)], 161 ± 23 b · min(-1), and 22.5 ± 11.1 minutes) than for DDR (119 ± 16 b · min(-1) [37% HRR], 138 ± 20 b · min(-1), and 11.2 ± 11.9 minutes) or for CE (126 ± 20 b · min(-1) [42% HRR], 144 ± 24 b · min(-1), and 14.2 ± 12.6 minutes). The RPE was significantly higher for GB (4.2 ± 1.5) and CE (3.8 ± 1.2) than for DDR (2.7 ± 1.3). Recovery HR (RecHR) (15 minutes postexercise) was significantly higher for GB (91 ± 14 b · min(-1)) than for DDR (80 ± 11 b · min(-1)) and neared significance vs. CE (84 ± 14 b · min(-1), p = 0.059). No difference in PkHR, RecHR, or minutes above THR was observed between DDR and CE. Session RPE was significantly higher for GB (4.6 ± 1.7) and CE (4.1 ± 1.6) than for DDR (2.8 ± 1.5). All modes elicited extended proportions of time above THR; GB: 75%, DDR: 37%, and CE: 47%. Results support that exergames are capable of eliciting physiological responses necessary for fitness improvements. Practitioners might consider exergames as periodic activity options for clients needing motivation to be regularly active.     

Role of four calcium transport proteins, encoded by nca-1, nca-2, nca-3, and cax, in maintaining intracellular calcium levels in Neurospora crassa

We have examined the distribution of calcium in Neurospora crassa and investigated the role of four predicted calcium transport proteins. The results of cell fractionation experiments showed 4% of cellular calcium in mitochondria, approximately 11% in a dense vacuolar fraction, 40% in an insoluble form that copurifies with microsomes, and 40% in a high-speed supernatant, presumably from large vacuoles that had broken. Strains lacking NCA-1, a SERCA-type Ca(2+)-ATPase, or NCA-3, a PMC-type Ca(2+)-ATPase, had no obvious defects in growth or distribution of calcium. A strain lacking NCA-2, which is also a PMC-type Ca(2+)-ATPase, grew slowly in normal medium and was unable to grow in high concentrations of calcium tolerated by the wild type. Furthermore, when grown in normal concentrations of calcium (0.68 mM), this strain accumulated 4- to 10-fold more calcium than other strains, elevated in all cell fractions. The data suggest that NCA-2 functions in the plasma membrane to pump calcium out of the cell. In this way, it resembles the PMC-type enzymes of animal cells, not the Pmc1p enzyme in Saccharomyces cerevisiae that resides in the vacuole. Strains lacking the cax gene, which encodes a Ca(2+)/H(+) exchange protein in vacuolar membranes, accumulate very little calcium in the dense vacuolar fraction but have normal levels of calcium in other fractions. The cax knockout strain has no other observable phenotypes. These data suggest that "the vacuole" is heterogeneous and that the dense vacuolar fraction contains an organelle that is dependent upon the CAX transporter for accumulation of calcium, while other components of the vacuolar system have multiple calcium transporters.     

A role for both conformational selection and induced fit in ligand binding by the LAO protein

Molecular recognition is determined by the structure and dynamics of both a protein and its ligand, but it is difficult to directly assess the role of each of these players. In this study, we use Markov State Models (MSMs) built from atomistic simulations to elucidate the mechanism by which the Lysine-, Arginine-, Ornithine-binding (LAO) protein binds to its ligand. We show that our model can predict the bound state, binding free energy, and association rate with reasonable accuracy and then use the model to dissect the binding mechanism. In the past, this binding event has often been assumed to occur via an induced fit mechanism because the protein's binding site is completely closed in the bound state, making it impossible for the ligand to enter the binding site after the protein has adopted the closed conformation. More complex mechanisms have also been hypothesized, but these have remained controversial. Here, we are able to directly observe roles for both the conformational selection and induced fit mechanisms in LAO binding. First, the LAO protein tends to form a partially closed encounter complex via conformational selection (that is, the apo protein can sample this state), though the induced fit mechanism can also play a role here. Then, interactions with the ligand can induce a transition to the bound state. Based on these results, we propose that MSMs built from atomistic simulations may be a powerful way of dissecting ligand-binding mechanisms and may eventually facilitate a deeper understanding of allostery as well as the prediction of new protein-ligand interactions, an important step in drug discovery.