A Refined Model for the Structure of Acireductone Dioxygenase from Klebsiella ATCC 8724 Incorporating Residual Dipolar Couplings

Acireductone dioxygenase (ARD) from Klebsiella ATCC 8724 is a metalloenzyme that is capable of catalyzing different reactions with the same substrates (acireductone and O₂) depending upon the metal bound in the active site. A model for the solution structure of the paramagnetic Ni²⁺-containing ARD has been refined using residual dipolar couplings (RDCs) measured in two media. Additional dihedral restraints based on chemical shift (TALOS) were included in the refinement, and backbone structure in the vicinity of the active site was modeled from a crystallographic structure of the mouse homolog of ARD. The incorporation of residual dipolar couplings into the structural refinement alters the relative orientations of several structural features significantly, and improves local secondary structure determination. Comparisons between the solution structures obtained with and without RDCs are made, and structural similarities and differences between mouse and bacterial enzymes are described. Finally, the biological significance of these differences is considered.     

Facial bristle feather histology and morphology in New Zealand birds: Implications for function

Knowledge of structure in biology may help inform hypotheses about function. Little is known about the histological structure or the function of avian facial bristle feathers. Here we provide information on morphology and histology, with inferences for function, of bristles in five predominantly insectivorous birds from New Zealand. We chose species with differing ecologies, including: brown kiwi (Apteryx mantelli), morepork (Ninox novaezealandae), hihi (Notiomystis cincta), New Zealand robin (Petroica australis), and New Zealand fantail (Rhipidura fuliginosa). Average bristle length corrected for body size was similar across species. Bristles occurred in distinct groups on different parts of the head and upper rictal bristles were generally longest. The lower rictal bristles of the fantail were the longest possessed by that species and were long compared to bristles of other species. Kiwi were the only species with forehead bristles, similar in length to the upper rictal bristles of other species, and the lower rictal bristles of fantails. Herbst corpuscles (vibration and pressure sensitive mechanoreceptors) were found in association with bristle follicles in all species. Nocturnal and hole‐nesting birds had more heavily encapsulated corpuscles than diurnal open‐nesting species. Our results suggest that avian facial bristles generally have a tactile function in both nocturnal and diurnal species, perhaps playing a role in prey handling, gathering information during flight, navigating in nest cavities and on the ground at night and possibly in prey‐detection. These differing roles may help explain the observed differences in capsule thickness of the corpuscles. J. Morphol., 2011. © 2010 Wiley‐Liss, Inc.     

A fluorescence-based high-throughput assay for antimicrotubule drugs

With the advent of combinatorial chemistry and the extensive libraries of potential drugs produced from it, there is a growing need for rapid sensitive, high-throughput screening for drug potency. Microtubules are important targets for anticancer agents, and new antimicrotubule compounds are of continued interest in drug development. The in vitro potency of antimicrotubule drugs may be evaluated by measuring the extent of tubulin assembly. The extent of polymerization is proportional to the turbidity of the solution, which usually has been measured as apparent absorption. The turbidity method has inherent problems that hinder its adaptation to a high-throughput format, such as a requirement for high protein concentrations and a high coefficient of variation. We present here a high-throughput assay for antimicrotubule activity in which fluorescence is used to monitor microtubule assembly. Both assembly-inhibiting and assembly-promoting compounds can be evaluated. The assay is rapid and easy to perform, and the data are reliable, with good accuracy and reproducibility.     

Measurement of Reactive Oxygen Species in the Culture Media Using Acridan Lumigen PS-3 Assay

Reactive oxygen species (ROS) are generated continuously during aerobic metabolism. ROS are highly reactive molecules and in excessive amounts, can lead to protein and DNA oxidation, protein cross-linking, and cell death. Cell-culture models provide a valuable tool in understanding the mechanisms that lead to cell death. Accumulation of ROS within cells and/or their release into the culture media are highly cell type-specific. The ability to estimate ROS levels in the culture media is an important step in understanding the mechanisms contributing to disease processes. In this paper, we describe the optimization of a simple method to estimate ROS levels in the culture media using the Acridan Lumigen PS-3 reagent provided in the Amersham ECL Plus kit (GE Healthcare, UK). We have shown that the Acridan Lumigen PS-3 assay generates ROS-specific chemiluminescence in fresh as well as media stored at −20°C, in as little as 10–20 μl of samples. The method was able to detect the dose (of stimulants)- and time (acute and chronic)-dependent changes in ROS levels in media collected from various cell types. Our results suggest that the kit reagents, PBS buffer, and various media did not contribute significantly to the overall chemiluminescence generated in the assay; however, we suggest that the unused medium specific for each cell type should be used as blanks and final readings of test samples normalized against these readings. As this method uses commonly available laboratory equipment and commercially available reagents, we believe this assay is convenient, economical, and specific in estimating ROS released extracellularly into the culture media.     

Scale-Up of Mammalian Cell Culture using a New Multilayered Flask

A growing number of cell-based applications require large numbers of cells. Usage of single layer T-flasks, that are adequate during small-scale expansion, may become cumbersome, laborious and time-consuming when large numbers of cells are required. To address this need, the performance of a new multi-layered cell culture vessel to facilitate easy scale up of cells from single layered T-flasks will be discussed. The flasks tested are available in 3- and 5-layer format and enable culture and complete recovery of three and five times the number of cells respectively, compared to T-175 flasks. A key feature of the BD Multi-Flask is a mix/equilibration port that allows rapid in-vessel mixing as well as uniform distribution of cells and reagents within and between layers of each vessel and consistently produce cells that can be cultured in an environment that is congruent to T-175 flasks.The design of these Multi-Flasks also allows for convenient pipette access for adding reagents and cells directly into the flasks as well as efficient recovery of valuable cells and reagents and reduces risk of contamination due to pouring. For applications where pouring is preferred over pipetting, the design allows for minimal residual liquid retention so as to reduce wastage of valuable cells and reagents.     

A stochastic population model for Lepidium papilliferum (Brassicaceae), a rare desert ephemeral with a persistent seed bank

Population viability analysis (PVA) is a valuable tool for rare plant conservation, but PVA for plants with persistent seed banks is difficult without reliable information on seed bank processes. We modeled the population dynamics of the Snake River Plains ephemeral Lepidium papilliferum using data from an 11-yr artificial seed bank experiment to estimate age-specific vital rates for viability loss and germination. We related variation in postgermination demographic parameters to annual variation in precipitation patterns and used these relationships to construct a stochastic population model using precipitation driver variables. This enabled us to incorporate realistic levels of environmental variability into the model. A model incorporating best estimates for parameter values resulted in a mean trajectory for seed bank size that remained essentially stable through time, although there was a measurable risk of extinction over a 100-yr period for the study population under this scenario. Doubling the annual seed viability loss rate resulted in near-certain extinction, as did increasing first-year germination to 100%, showing the importance of the persistent seed bank. Interestingly, increasing environmental variance substantially decreased the risk of extinction, presumably because this plant relies on extremely good years to restock the persistent seed bank, while extremely bad years have little impact. If every year were average in this desert environment, the species could not persist. Simulated effects of livestock trampling resulted in greatly increased extinction risk, even over time frames as short as 15 years.