Analysis of human cytochrome P450 3A4 cooperativity: Construction and characterization of a site-directed mutant that displays hyperbolic steroid hydroxylation kinetics

Cytochrome P450 3A4 is generally considered to be the most important human drug-metabolizing enzyme and is known to catalyze the oxidation of a number of substrates in a cooperative manner. An allosteric mechanism is usually invoked to explain the cooperativity. Based on a structure–activity study from another laboratory using various effector–substrate combinations and on our own studies using site-directed mutagenesis and computer modeling of P450 3A4, the most likely location of effector binding is in the active site along with the substrate. Our study was designed to test this hypothesis by replacing residues Leu-211 and Asp-214 with the larger Phe and Glu, respectively. These residues were predicted to constitute a portion of the effector binding site, and the substitutions were designed to mimic the action of the effector by reducing the size of the active site. The L211F/D214E double mutant displayed an increased rate of testosterone and progesterone 6β-hydroxylation at low substrate concentrations and a decreased level of heterotropic stimulation elicited by α-naphthoflavone. Kinetic analyses of the double mutant revealed the absence of homotropic cooperativity with either steroid substrate. At low substrate concentrations the steroid 6β-hydroxylase activity of the wild-type enzyme was stimulated by a second steroid, whereas L211F/D214E displayed simple substrate inhibition. To analyze L211F/D214E at a more mechanistic level, spectral binding studies were carried out. Testosterone binding by the wild-type enzyme displayed homotropic cooperativity, whereas substrate binding by L211F/D214E displayed hyperbolic behavior.

Construction of a high-affinity receptor site for dihydropyridine agonists and antagonists by single amino acid substitutions in a non-l-type Ca2+ channel

The activity of l-type Ca2+ channels is increased by dihydropyridine (DHP) agonists and inhibited by DHP antagonists, which are widely used in the therapy of cardiovascular disease. These drugs bind to the pore-forming α1 subunits of l-type Ca2+ channels. To define the minimal requirements for DHP binding and action, we constructed a high-affinity DHP receptor site by substituting a total of nine amino acid residues from DHP-sensitive l-type α1 subunits into the S5 and S6 transmembrane segments of domain III and the S6 transmembrane segment of domain IV of the DHP-insensitive P/Q-type α1A subunit. The resulting chimeric α1A/DHPS subunit bound DHP antagonists with high affinity in radioligand binding assays and was inhibited by DHP antagonists with high affinity in voltage clamp experiments. Substitution of these nine amino acid residues yielded 86% of the binding energy of the l-type α1C subunit and 92% of the binding energy of the l-type α1S subunit for the high-affinity DHP antagonist PN200–110. The activity of chimeric Ca2+ channels containing α1A/DHPS was increased 3.5 ± 0.7-fold by the DHP agonist (−)Bay K8644. The effect of this agonist was stereoselective as in l-type Ca2+ channels since (+) Bay K8644 inhibited the activity of α1A/DHPS. The results show conclusively that DHP agonists and antagonists bind to a single receptor site at which they have opposite effects on Ca2+ channel activity. This site contains essential components from both domains III and IV, consistent with a domain interface model for binding and allosteric modulation of Ca2+ channel activity by DHPs.

Construction and characterization of an azurin analog for the purple copper site in cytochrome c oxidase.

A protein analog of a purple copper center has been constructed from a recombinant blue copper protein (Pseudomonas aeruginosa azurin) by replacing the loop containing the three ligands to the blue copper center with the corresponding loop of the CuA center in cytochrome c oxidase (COX) from Paracoccus denitrificans. The electronic absorption in the UV and visible region (UV-vis) and electron paramagnetic resonance (EPR) spectra of this analog are remarkably similar to those of the native CuA center in COX from Paracoccus denitrificans. The above spectra can be obtained upon addition of a mixture of Cu2+ and Cu+. Addition of Cu2+ only results in a UV-vis spectrum consisting of absorptions from both a purple copper center and a blue copper center. This spectrum can be converted to the spectrum of a pure purple copper by a prolonged incubation in the air, or by addition of excess ascorbate. The azurin mutant reported here is an example of an engineered purple copper center with the A480/A530 ratio greater than 1 and with no detectable hyperfines, similar to those of the CuA sites in COX of bovine heart and of Paracoccus denitrificans.

In Situ Bioremediation Methods for Petroleum Pollutants in Soil: A Training Manual for Construction Companies

Construction companies are interested in adopting environmental methods, such as in situ bioremediation, when conducting remediation at petroleum contaminated sites. This interest is due to both the cost benefits associated with in situ bioremediation methods and the environmental movement. This project creates a comprehensive manual comprised of information about site investigations, treatability studies, design, and implementation of in situ bioremediation at petroleum contaminated sites. The training manual also provides information about regulatory requirements and permitting for petroleum contaminated sites.

Implementation Feasibility of a Digital Nervous System for the Construction Industry: For Efficient and Effective Information Management across the Project Lifecycle

The construction industry has long been considered as highly fragmented and non-collaborative industry. This fragmentation sprouted from complex and unstructured traditional coordination processes and information exchanges amongst all parties involved in a construction project. This nature coupled with risk and uncertainty has pushed clients and their supply chain to search for new ways of improving their business process to deliver better quality and high performing product. This research will closely investigate the need to implement a Digital Nervous System (DNS), analogous to a biological nervous system, on the flow and management of digital information across the project lifecycle. This will be through direct examination of the key processes and information produced in a construction project and how a DNS can provide a well-integrated flow of digital information throughout the project lifecycle. This research will also investigate how a DNS can create a tight digital feedback loop that enables the organisation to sense, react and adapt to changing project conditions. A Digital Nervous System is a digital infrastructure that provides a well-integrated flow of digital information to the right part of the organisation at the right time. It provides the organisation with the relevant and up-to-date information it needs, for critical project issues, to aid in near real-time decision-making. Previous literature review and survey questionnaires were used in this research to collect and analyse data about information management problems of the industry – e.g. disruption and discontinuity of digital information flow due to interoperability issues, disintegration/fragmentation of the adopted digital solutions and paper-based transactions. Results analysis revealed efficient and effective information management requires the creation and implementation of a DNS.

Construction of a precise early Eocene orbital cyclostratigraphy for DSDP Site 80-550 from the Goban Spur, North Atlantic

We constructed a precise early Eocene orbital cyclostratigraphy for DSDP Site 550 (Leg 80, Goban Spur, North Atlantic) utilizing precession related cycles as represented in a high resolution X-Ray Fluorescence based Barium core log. Based on counting of those cycles, we constrain the exact timing of two volcanic ash layers in Site 550 which correlate to ashes +19 and -17 of the Fur Formation in Denmark. The ashes, relative to the onset of the Paleocene/Eocene Thermal Maximum (PETM), are offset by 862 kyr and 672 kyr, respectively. When combined with published absolute ages for ash -17, the absolute age for the onset of the PETM is consistent with astronomically calibrated ages. Using the current absolute age of 28.02 Ma for the Fish Canyon Tuff (FCT) standard for calibrating the absolute age of ash -17 is consistent with tuning option 2 in the astronomically calibrated Paleocene time scale of Westerhold et al. (2008) [Westerhold, T., Röhl, U., Raffi, I., Fornaciari, E., Monechi, S., Reale, V., Bowles, J., and Evans, H.F., 2008, Astronomical calibration of the Paleocene time: Palaeogeography, Palaeoclimatology, Palaeoecology, v. 257, p. 377-403]. Using the recently recalibrated absolute age of 28.201 Ma for the FCT standard is consistent with tuning option 3 in the astronomically calibrated Paleocene time scale. The new results do not support the existence of any additional 405-kyr cycle in the early Paleocene astronomically tuned time scale.

Herbert Hoover national historic site, interpretive prospectus (1971) B1; Natural resources management plan (1980) B2; Cultural resources management plan (1981) B3; Annual statement for interpretation and visitor services B4; Snowmobile use, environmental assessment (EA) : environmental impact statement.

Mode of access: Internet.

Energy conservation site visit report : toward more effective energy management /

S/N 041-018-00100-0

Recovery of landfill gas at Mountain View : engineering site study : a current report on solid waste management /

Mode of access: Internet.

Radioactive waste management : a bibliography of publicly available literature pertaining to the USAEC's Savannah River, S.C., production site /

Mode of access: Internet.

Radioactive waste management : a bibliography of publicly available literature pertaining to the USAEC's Hanford, Washington Production Site /

"August 1973."

Evaluation of the refuse management system at the Jersey City operation breakthrough site /

Mode of access: Internet.

The book of duck decoys, their construction, management, and history

Mode of access: Internet.