Correspondence - Attie Bostick Feb. 23, 1934 - Wade Bostick

A correspondence from Attie T. Bostick. Includes handwritten note to Mrs. H.L. Packard and a partial letter from Wade Bostick.https://digitalcommons.gardner-webb.edu/baptist-historical-collections-bostick-family-missions/1019/thumbnail.jp

Use of protein immobilization to measure cytochrome P450 conduction and metabolism kinetics

Cytochrome P450s (P450s) are a large family (\u3e11,000) of heme thiolated proteins that are responsible for ~ 75% of the metabolism of pharmaceuticals on the market. Understanding P450 mediated metabolism is crucial for accurate in vitro predictions of drug metabolism. P450 protein-protein interactions have been shown to alter enzyme catalytic activity. Furthermore, these interactions are isoform specific, and can elicit activation, inhibition, or no effect on enzymatic activity. Studies show these effects are also dependent on the protein binding partner cytochrome P450 reductase (CPR), and the order of protein addition to purified reconstituted enzyme systems. In the current work, we use controlled immobilization of P450s to a gold surface to gain a better understanding of P450-P450 interactions between three key drug-metabolizing isoforms (CYP2C9, CYP3A4, and CYP2D6). Molecular modeling was used to assess the favorability of homo/heteromeric P450 complex formation. P450 complex formation in vitro was analyzed in real-time utilizing surface plasmon resonance (SPR). Lastly, the effects of P450 complex formation were investigated utilizing our immobilized platform and reconstituted enzyme systems.;Molecular modeling shows favorable binding of CYP2C9-CPR, CYP2C9-CYP2D6, CYP2C9-CYP2C9, and CYP2C9-CYP3A4 in rank order. KD values obtained via SPR show strong binding, in the nanomolar range, of the above pairs, with CYP2D6 yielding the lowest KD, followed by CYP2C9, CPR, and CYP3A4. Metabolic incubations show immobilized CYP2C9 metabolism was activated by homomeric complex formation. CYP2C9 metabolism was not affected by the presence of CYP3A4 with saturating CPR concentrations. CYP2C9 metabolism was activated by CYP2D6 in solution, but inhibited when CYP2C9 was immobilized, both at saturating and sub-saturating CPR concentrations. Order of addition of proteins (CYP2C9, CYP2D6, CYP3A4, and CPR) influenced magnitude of inhibition for CYP3A4, but not CYP2D6. These results indicate isoform specific P450 interactions and effects on P450 mediated-metabolism. These findings are important in evaluating how in vitro results are obtained for measuring P450 kinetics, and provide a better mechanistic understanding of P450-P450 interactions to allow for better prediction of in vivo metabolism from in vitro data.;We also demonstrate that gold nanopillars, functionalized with an organic self-assembled monolayer, can be used to measure the electrical conductance properties of immobilized P450s without aggregation. Given that transfer of the 1st electron to the P450 heme group acts as the gating step for the catalytic cycle, understanding electron transfer in P450s could shed light on metabolism kinetics. Conductance measurements of nanopillars with immobilized CYP2C9 using conducting probe atomic force microscopy demonstrate that a correlation exists between the energy barrier height between hopping sites and CYP2C9 metabolic activity. Measurements performed as a function of tip force indicate that, when subjected to a large force, the protein is more stable in the presence of a substrate. This agrees with the hypothesis that substrate entry into the active site helps to stabilize the enzyme.;The relative distance between hopping sites also increases with increasing force, possibly because protein functional groups responsible for electron transport depend on the structure of the protein. The inhibitor sulfaphenazole, in addition to the previously studied aniline, increased the barrier height for electron transfer and thereby makes CYP2C9 reduction more difficult and inhibits metabolism. This suggests that P450 Type II ligands may decrease the ease of electron transport processes in the enzyme, in addition to occupying the active site. These findings further our understanding of how P450 metabolism is mediated through substrates, and provides an important technological advancement for studying P450s that avoids complications found in current methodologies. These two studies demonstrate the ability of an immobilized P450 platform to provide information on protein-protein interactions, substrate protein interactions, and atypical enzyme kinetics

Electroreduction and photometric detection of low-level uranium in aqueous Purex solutions. Consolidated Fuel-Reprocessing Program

During proper operation of the Purex process for the recovery of uranium and plutonium from spent reactor fuel, there are only trace levels of uranium in the aqueous waste. In the event of an upset in the extraction columns the aqueous waste stream would give the first indication of breakthrough. From the standpoint of process control it would be desirable to have an in-line, real-time sensor for uranium in the aqueous waste stream. It was toward this end that this investigation was undertaken. The measurement technique that seems to provide the most sensitive method without addition of reagents appears to be the electrochemical reduction of UO{sub 2}{sup 2+} to U(IV) followed by spectral measurement. The electrochemical reduction to U(IV) increases the sensitivity by a factor of three to five and shifts the measurement wavelength to a spectral area (647 nm and 1075 nm) unaffected by fission products. Using the proposed analysis sequence it is possible to determine uranium at a level of 0.2 g/L in the presence of relatively high spectral background. This report details the electrochemical reduction of U(VI) in nitric acid solutions (0.5 M to 2.0 M) with platinum-vitreous carbon electrodes and examines the spectral behavior of U(IV) as a function of nitric acid concentration

Value of Private Lands Managed for Wild Northern Bobwhites in the Deep South

Successful northern bobwhite (Colinus virginiaus) management creating huntable bobwhite populations in the southeastern United States is largely practiced on private lands. These properties not only support high density bobwhite populations, they also support biodiversity including many declining or endangered species associated with frequently-burned pine savannas. The private land model has proven sustainable over centuries and has recently increased with \u3e20,000 ha of wild bobwhite lands added in Georgia, Florida, and South Carolina since 2000. The NBCI recognizes private lands are critical to restoration of bobwhites but, despite their conservation value, no studies have quantified areas under wild bobwhite management. We mapped 259 properties totaling ~ 345,614 ha with wild bobwhite management principally in Georgia, Florida, Alabama, and South Carolina, and are completing mapping in Mississippi, Virginia, and North Carolina. Our survey data found management actions were consistent across these properties including maintaining open pine canopy, reducing hardwoods in upland areas, prescribed fire on areas of appropriate sizes and distribution, year- round supplemental feeding, nest predator management, and conservative harvest rates. Adoption of these management practices are a result of long-term research demonstrating their efficacy. Density of bobwhites on a property was related to landscape composition with lower densities on more fragmented sites. Properties in core areas surrounded by other managed properties often achieved bobwhite densities of 5–8 quail/ha. Bobwhite densities on smaller isolated properties densities were ~ 2.5 quail/ha during fall. Aspects of this successful management model may be useful to other private lands as well as public management areas focused on northern bobwhite

Effects of Supplemental Feeding on Breeding Season Home Ranges and Resource Selection of Northern Bobwhites

Providing supplemental food resources for northern bobwhite (Colinus virginanus) has been a common management practice for decades, but its impact on bobwhite home ranges sizes and space use has been debated. Between 2001 and 2007, we established a 397 ha fed study area on Tall Timbers Research Station, which received grain sorghum biweekly along a 19.5 km feed trail. An adjacent 465 ha area was treated as a control with no supplemental food resources. Radio-tagged wild bobwhites were located 3 – 5 times each week throughout the breeding season and we used these locations to calculate home ranges during early breeding season (15 April – 30 June) and late breeding season (1 July – 1 October). We also determined second and third order selection of a 10-m buffer area surrounding the supplemental feed trail using compositional analyses. In total, 552 and 286 bobwhite home ranges were calculated for early and late breeding seasons, respectively. We observed significantly smaller early breeding season home ranges on the fed area (x̄ = 12.3 ha, 95% CL ± 0.6) relative to the control (x̄ = 17.4 ha ± 1.9). Average length of feed trail within home ranges was 583 m and 710 m for early and late breeding season home ranges, respectively. The feed trail buffer area was 3 to 5% of all home ranges. Home range placement was not random with high selection preference for the feed trail buffer area. However, there was a low selection preference for the feed trail buffer area within home ranges. Average distances to the feed trail for bobwhite locations (x̄ = 64.4 m ± 16.1) was similar to random locations (x̄ = 74.4 m ± 16.9). The effect of supplemental feeding on bobwhite home ranges size and resource use was greatest during the early breeding season when food and useable habitat were likely lower in availability. On our study area, supplemental food resources distributed along a feed trail impacted bobwhites during a critical transition period from the spring prescribed burning season to the early breeding season

Dehydration and ionic conductance quantization in nanopores

There has been tremendous experimental progress in the last decade in identifying the structure and function of biological pores (ion channels) and fabricating synthetic pores. Despite this progress, many questions still remain about the mechanisms and universal features of ionic transport in these systems. In this paper, we examine the use of nanopores to probe ion transport and to construct functional nanoscale devices. Specifically, we focus on the newly predicted phenomenon of quantized ionic conductance in nanopores as a function of the effective pore radius - a prediction that yields a particularly transparent way to probe the contribution of dehydration to ionic transport. We study the role of ionic species in the formation of hydration layers inside and outside of pores. We find that the ion type plays only a minor role in the radial positions of the predicted steps in the ion conductance. However, ions with higher valency form stronger hydration shells, and thus, provide even more pronounced, and therefore, more easily detected, drops in the ionic current. Measuring this phenomenon directly, or from the resulting noise, with synthetic nanopores would provide evidence of the deviation from macroscopic (continuum) dielectric behavior due to microscopic features at the nanoscale and may shed light on the behavior of ions in more complex biological channels.Comment: 13 pages, 10 figure

Habitat Selection by Northern Bobwhite Broods in Pine Savanna Ecosystems

Habitat for northern bobwhite (Colinus virginianus) broods is a critical component of bobwhite management. Research within pine (Pinus spp.) savannas has provided contradictory results regarding the value of macro-habitats with studies demonstrating selection for annually-disked fallow fields and others showing avoidance of fields and selection for burned pine savannas. Field establishment (up to 30% of a property) is a published management recommendation for bobwhites in pine savannas but there are significant annual costs with fallow-field management; information on factors that influence habitat selection by broods can improve management recommendations and facilitate weighing costs/benefits. We examined 2nd and 3rd order habitat selection by 466 broods on 3 sites during 1999–2009. All sites had similar macro-habitats (e.g., pine savanna, fallow fields, hardwood drains) but differed in soil characteristics and species composition of ground vegetation. Annually-disked fields were preferred by broods in most years on sites with predominantly grass and hardwood scrub ground vegetation. Rainfall mediated use of hardwood drains and burned upland pine savannas; hardwood drains were used more during droughts whereas burned pine savannas were used more with increased rainfall. Burned upland pine savanna was preferred on higher fertility sites in 9 of 10 years at the 3rd order level, fields were avoided or used according to availability in 8 of 10 years, and drains were avoided. Managers should consider how soil, weather, and vegetation community in pine savannas influences habitat use by bobwhite broods when identifying the value of different macro-habitats. Field establishment may or may not provide brood habitat depending on site

Wax Point Determinations Using Acoustic Resonance Spectroscopy

The thermodynamic characterization of the wax point of a given crude is essential in order to maintain flow conditions that prevent plugging of undersea pipelines. This report summarizes the efforts made towards applying an Acoustic Cavity Resonance Spectrometer (ACRS) to the determination of pressures and temperatures at which wax precipitates from crude. Phillips Petroleum Company, Inc., the CRADA participant, supplied the ACRS. The instrumentation was shipped to Dr. Thomas Schmidt of ORNL, the CRADA contractor, in May 2000 after preliminary software development performed under the guidance of Dr. Samuel Colgate and Dr. Evan House of the University of Florida, Gainesville, Fl. Upon receipt it became apparent that a number of modifications still needed to be made before the ACRS could be precisely and safely used for wax point measurements. This report reviews the sequence of alterations made to the ACRS, as well as defines the possible applications of the instrumentation once the modifications have been completed. The purpose of this Cooperative Research and Development Agreement (CRADA) between Phillips Petroleum Company, Inc. (Participant) and Lockheed Martin Energy Research Corporation (Contractor) was the measurement of the formation of solids in crude oils and petroleum products that are commonly transported through pipelines. This information is essential in the proper design, operation and maintenance of the petroleum pipeline system in the United States. Recently, new petroleum discoveries in the Gulf of Mexico have shown that there is a potential for plugging of undersea pipeline because of the precipitation of wax. It is important that the wax points of the expected crude oils be well characterized so that the production facilities for these new wells are capable of properly transporting the expected production. The goal of this work is to perform measurements of solids formation in crude oils and petroleum products supplied by the Participant. It is anticipated that these data will be used in the design of new production facilities and in the development of thermodynamic models that describe the behavior of wax-saturated petroleum

Selection of Single-Stranded DNA Molecular Recognition Elements against Exotoxin A Using a Novel Decoy-SELEX Method and Sensitive Detection of Exotoxin A in Human Serum

Exotoxin A is one of the virulence factors of Pseudomonas aeruginosa, a bacterium that can cause infections resulting in adverse health outcomes and increased burden to health care systems. Current methods of diagnosing P. aeruginosa infections are time consuming and can require significant preparation of patient samples. This study utilized a novel variation of the Systematic Evolution of Ligand by Exponential Enrichment, Decoy-SELEX, to identify an Exotoxin A specific single-stranded DNA (ssDNA) molecular recognition element (MRE). Its emphasis is on increasing stringency in directing binding toward free target of interest and at the same time decreasing binding toward negative targets. A ssDNA MRE with specificity and affinity was identified after fourteen rounds of Decoy-SELEX. Utilizing surface plasmon resonance measurements, the determined equilibrium dissociation constant of the MRE is between 4.2 µM and 4.5 µM, and is highly selective for Exotoxin A over negative targets. A ssDNA MRE modified sandwich enzyme-linked immunosorbent assay (ELISA) has been developed and achieved sensitive detection of Exotoxin A at nanomolar concentrations in human serum. This study has demonstrated the proof-of-principle of using a ssDNA MRE as a clinical diagnostic tool