Conformationally Gated Electron Transfer in Nitrogenase. Isolation, Purification, and Characterization of Nitrogenase From Gluconacetobacter diazotrophicus
Cedric P. Owens and Faik A. Tezcan
Nitrogenase is a complex, bacterial enzyme that catalyzes the ATP-dependent reduction of dinitrogen (N2) to ammonia (NH3). In its most prevalent form, it consists of two proteins, the catalytic molybdenum-iron protein (MoFeP) and its specific reductase, the iron protein (FeP). A defining feature of nitrogenase is that electron and proton transfer processes linked to substrate reduction are synchronized by conformational changes driven by ATP-dependent FeP-MoFeP interactions. Yet, despite extensice crystallographic, spectroscopic, and biochemical information on nitrogenase, the structural basis of the ATP-dependent synchronization mechanism is not understood in detail. In this chapter, we summarize some of our efforts toward obtaining such an understanding.
Experimental investigations of the structure-function relationships in nitrogenase are challenged by the fact that it cannot be readily expressed herterlogously in non-diazotrophic bacteria, and the purification protocols for nitrogenase are only known for a small number of diazotrophic organisms. Here, we present methods for purifying and characterizing nitrogenase from a new model organism, Gluconacetobacter diazotrophicus. We also describe procedures for observing redox-dependent conformational changes in G. diazotrophicus nitrogenase by X-ray crystallography and electron paramagnetic resonance spectroscopy, which have provided new insights into the redox-dependent conformational gating processes in nitrogenase.
Frederick J. Ehlert, K. J. Pak, and Michael T. Griffin
Muscarinic agonists and antagonists are used to treat a handful of gastrointestinal (GI) conditions associated with impaired salivary secretion or altered motility of GI smooth muscle. With regard to exocrine secretion, the major muscarinic receptor expressed in salivary, gastric, and pancreatic glands is the M3 with a small contribution of the M1 receptor. In GI smooth muscle, the major muscarinic receptors expressed are the M2 and M3 with the M2 outnumbering the M3 by a ratio of at least four to one. The antagonism of both smooth muscle contraction and exocrine secretion is usually consistent with an M3 receptor mechanism despite the major presence of the M2 receptor in smooth muscle. These results are consistent with the conditional role of the M2 receptor in smooth muscle. That is, the contractile role of the M2 receptor depends on that of the M3 so that antagonism of the M3 receptor eliminates the response of the M2. The physiological roles of muscarinic receptors in the GI tract are consistent with their known signaling mechanisms. Some so-called tissue-selective M3 antagonists may owe their selectivity to a highly potent interaction with a nonmuscarinic receptor target.
Douglas S. Fudge
"Hagfish slime threads form the fibrous fraction of the defensive slime of hagfishes. The threads develop within specialized cells within the slime glands and consist of a dense bundle of the cytoskeletal elements known as intermediate filaments. The threads leave the hagfish's body in a condensed state, but quickly unravel into fine protein strands that are more than 10 cm long..."
Robert E. Shadwick, Laurenne L. Schiller, and Douglas S. Fudge
Tunas are well known for specialized anatomical and physiological features that correlate to their exceptionally active lifestyle and ability to migrate over long distances. Foremost are a high degree of body streamling, a lunate tail fin with high aspect ratio, a relatively "stiff-body" swimming style, elevated core body temperature, medially and anteriorly located aerobic red muscle, a large heart, and a high metabolic rate. This chapter will discuss the state of knowledge of these properties and review the importance of the muscle and tendon anatomy, muscle activation patterns and contractile properties, and the effect of elevated temperature on muscle power output. We also present an assessment of tuna swimming performance based on laboratory and field measurements, and summarize current knowledge of tuna migration patterns. Finally, the use of tunas in aquaculture and its future potential is discussed.
Analyzing Black Cloud Dynamics over Cairo, Nile Delta Region and Alexandria using Aerosols and Water Vapor Data
Hesham el-Askary, Anup K. Prasad, George Kallos, Mohamed El Raey, and Menas Kafatos
Cairo is the largest city of Africa and one of the world’s megacities, with a population of more than 20 million people and containing more than one third of the national industry. It is a rapidly expanding city which leads to many associated environmental problems. As a result, it is also one of the most air polluted megacities in the world (Molina and Molina, 2004). It suffers from high ambient concentrations of atmospheric pollutants including particulates (PM), carbon monoxide, nitrogen oxides, ozone and sulfur dioxide (Abu-Allaban et al., 2007, Abu-Allaban et al., 2002, El-Metwally et al., 2008). The pollution phenomenon locally known as “Black cloud” over Cairo has been attributed to many reasons among which are biomass burning, local emission and long range transport during the fall season.Several studies have been conducted to address and discuss the forth mentioned reasons for the increased pollution levels over Cairo and the greater Delta region using ground-based and satellite air quality data as compared to other megacities.
Anup K. Prasad, Hesham el-Askary, Ghassem R. Asrar, Menas Kafatos, and Ashok Jaswal
The Himalayan and Tibet Glaciers, that are among the largest bodies of ice and fresh water resource outside of the polar ice caps, face a significant threat of accelerated meltdown in coming decades due to climate variability and change. The rate of retreat of these glaciers and changes in their terminus (frontal dynamics) is highly variable across the Himalayan range. These large freshwater sources are critical to human activities for food production, human consumption and a whole host of other applications, especially over the Indo-Gangetic (IG) plains. They are also situated in a geo-politically sensitive area surrounded by China, India, Pakistan, Nepal and Bhutan where more than a billion people depend on them. The major rivers of the Asian continent such as the Ganga (also known as Ganges), Brahmaputra, Indus, Yamuna, Sutluj etc., originate and pass through these regions and they have greater importance due to their multi-use downstream: hydro power, agriculture, aquaculture, flood control, and as a freshwater resource. Recent studies over the Himalayan Glaciers using ground-based and space-based observations, and computer models indicate a long-term trend of climate variability and change that may accelerate melting of the Himalayan Glaciers.
Jae Kyu Cho, Zhiyong Meng, L. Andrew Lyon, and Victor Breedveld
Due to the tunability of their softness and volume as a function of temperature, poly(N-isopropylacrylamide) (pNIPAm) hydrogel particles have emerged as a model system for soft colloidal spheres. By introducing AAc as comonomer, one can also tune the particle volume via pH. We report on the phase behavior of these stimuli-responsive colloids as measured with a microdialysis cell. This device, which integrates microfluidics with Particle Tracking Video-microscopy allows for simple and quick investigation of the phase behavior of suspensions the soft colloidal hydrogel as a function of pH as well as its packing density. In particular, we demonstrate the existence of an unusually broad liquid/crystal coexistence region as a function of effective particle volume fraction. Additionally, we reveal that nonequilibrium jammed states can be created in the coexistence region upon sudden large changes of pH. The phase diagram is indicative of complex interparticle interactions with weakly attractive components.
Scott D. Bridgham, J. Patrick Megonigal, Norman B. Bliss, and Carl Trettin
North America is currently a net source of CO2 to the atmosphere, contributing to the global buildup of greenhouse gases in the atmosphere and associated changes in the Earth's climate. In 2003, North America emitted nearly two billion metric tonnes of carbon to the atmosphere as CO2. North America's fossil-fuel emissions in 2003 (1856 million metric tonnes of carbon ±10% with 95% certainty) were 27% of global emissions. Approximately 85% of those emissions were from the USA, 9% from Canada, and 6% from Mexico. The combustion of fossil fuels for commercial energy (primarily electricity) is the single largest contributor, accounting for approximately 42% of North American fossil emissions in 2003. Transportation is the second largest, accounting for 31% of total emissions. There are also globally important carbon sinks in North America. In 2003, growing vegetation in North America removed approximately 500 million tonnes of carbon per year (±50%) from the atmosphere and stored it as plant material and soil organic matter. This land sink is equivalent to approximately 30% of the fossil-fuel emissions from North America. The imbalance between the fossil-fuel source and the sink on land is a net release to the atmosphere of 1350 million metric tonnes of carbon year (±25%). Approximately 50% of North America's terrestrial sink is due to the regrowth of forests in the USA on former agricultural land that was last cultivated decades ago, and on timberland recovering from harvest. Other sinks are relatively small and not well quantified with uncertainties of 100% or more. The future of the North American terrestrial sink is also highly uncertain. The contribution of forest regrowth is expected to decline as the maturing forests grow more slowly and take up less CO2 from the atmosphere. However, the response of regrowing forests and other sinks to climatic changes and CO2 concentration in the atmosphere is highly uncertain. The large difference between current sources and sinks and the expectation that the difference could become large if the growth of fossil-fuel emissions continues and land sinks decline suggest that addressing imbalances in the North American carbon budget will likely require actions focused on reducing fossil-fuel emissions. Options to enhance sinks (growing forests or sequestering carbon in agricultural soils) can contribute, but enhancing sinks alone is likely insufficient to deal with either the current or future imbalance. Options to reduce emissions include efficiency improvement, fuel switching, and technologies such as carbon capture and geological storage. Implementing these options will likely require an array of policy instruments at local, regional, national, and international levels, ranging from the encouragement of voluntary actions to economic incentives, tradable emissions permits, and regulations. Meeting the demand for information by decision makers will likely require new modes of research characterized by close collaboration between scientists and carbon management stakeholders.
Daoji Gan and L. Andrew Lyon
Thermoresponsive poly(N-isopropylacrylamide) (pNIPAm) core/shell particles bearing primary amine groups in either core or shell were prepared via two-stage, free radical precipitation polymerization, using 2-aminoethyl methacrylate (AEMA) as a comonomer. The amine groups were then used to initiate ring-opening polymerization of γ-benzyl L-glutamate N-carboxyanhydride (BLG-NCA), producing poly(γ-benzyl L-glutamate) (PBLG) side chains covalently anchored to the particles. Photon Correlation Spectroscopy (PCS) and 1H NMR were employed to characterize these particles. A shift of phase transition to a lower temperature and an increase in particle des welling volume ratios were observed as a result of grafting hydrophobic PBLG chains to the particles. Further studies by 1H NMR in different solvents indicate that the PBLG chains grafted from the particle shell phase separate on the pNIPAm networks in aqueous media but remain well solvated in DMSO. Together, these results suggest that both core- and shell-grafted architectures can be synthesized with equal ease, and that the particle structure and colloidal behavior can be manipulated by tuning the relative solubility of the network and graft portions of the particle.
J. Michael Carthcart, L. Andrew Lyon, Marcus Weck, and Robert D. Bock
In this paper we present results from our research into the use of microgel-based photonic crystals in an optical tagging application. The basis for this research is the phenomena of self-assembly of hydrogel nano- and microparticles (i.e., microgels) into colloidal crystal Bragg reflectors. Previous research has demonstrated the assembly of Bragg structures that are sensitive in the visible spectral region. This current research focuses on the extension of this process into the infrared regime and the use of these infrared-sensitive structures in the creation of an optical tag. In particular, the research effort emphasizes two primary areas: the development of nanoparticles that are infrared-sensitive and the casting of thin films comprised of these particles. We will also present theoretical data on the optical and physical characteristics of thin films comprised of these particles. This paper will present an overview of the program, outline the processes and issues addressed during our initial efforts in creating these infrared sensitive structures and present a summary of the computational results based on the theoretical analyses.
Jennifer L. Funk and Manuel T. Lerdau
This chapter focuses on canopy photosynthesis.
Frederick J. Ehlert, Elizabeth A. Thomas, Edward H. Gersten, and Michael T. Griffin
Muscarinic acetylcholine M2 and M3 receptor subtypes are coexpressed in many types of smooth muscle including gastrointestinal smooth muscle, urinary bladder and vascular and airway tissue. Activation of M3 receptors, via the G protein Gq, results in increased polyphosphoinositide hydrolysis, release of Ca2+ ions from the sarcoplasmic reticulum and consequently causes contraction. Quantitation of the relative expression of M2 and M3 receptors has shown that the proportion of M2 receptors often predominates over the M3 receptor population by 4:1 or more. Although it is established that M2 receptors preferentially link, via a pertussis-toxin-sensitive G protein Gi, to inhibition of adenylate cyclase activity, relatively little is known concerning the physiological role of the M2 receptor population. In this review, Richard Eglen and colleagues discuss recent data concerning the possible role(s) of muscarinic receptor subtypes in smooth muscle and appraise the pharmacological methods for dissecting the function of muscarinic receptor subtypes in tissues co-expressing multiple receptors.
Mechanisms of Multiple Cellular Adaptation Processes in Clonal Cell Lines During Chronic Opiate Treatment
Ping-Yee Law, Michael T. Griffin, and Horace H. Loh
Among the various in vitro models for studies of opiate action, the clonal cell line, neuroblastoma x glioma NG 108-15 hybrid cells, represents the simplest model of brain origin in which the molecular basis of opiate action is being investigated. The hybrid cells possess many neuronal properties, such as having excitable membrane containing light and dense core vesicles; also the hybrid cells can be synapsed onto striatal muscle cells (Hamprecht 1977).