1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine as a substrate of cytochrome P450 2D6: allosteric effects of NADPH-cytochrome P450 reductase

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a neurotoxin that produces Parkinsonism symptoms in man, has been examined as a substrate of recombinant human cytochrome P450 2D6. When cumene hydroperoxide is used as an oxygen and electron donor, a single product is formed, identified as 4-phenyl-1,2,3,6-tetrahydropyridine. The K(m) for formation of this product (130 microM) is in agreement with the dissociation constants for MPTP binding to the enzyme determined by optical and nuclear magnetic resonance (NMR) spectroscopy. When the reaction is carried out with nicotinamide adenine dinucleotide phosphate (reduced) (NADPH) and recombinant human NADPH-cytochrome P450 reductase, a second product, identified as 1-methyl-4-(4'-hydroxyphenyl)-1,2,3,6-tetrahydropyridine, is formed in addition to 4-phenyl-1,2,3,6-tetrahydropyridine. The K(m) values for formation of these two products are 19 microM and 120 microM, respectively. Paramagnetic relaxation experiments have been used to measure distances between the protons of bound MPTP and the heme iron, and these have been used to construct models for the position and orientation of MPTP in the active site. For the cytochrome alone, a single mode of binding was observed, with the N-methyl close to the heme iron in a position appropriate for the observed N-demethylation reaction. In the presence of the reductase, the data were not consistent with a single mode of binding but could be explained by the existence of two alternative orientations of MPTP in the active site. One of these, characterized by a dissociation constant of 150 microM, is essentially identical to that observed in the absence of the reductase. In the second, which has a K(d) of 25 microM, the MPTP is oriented so that the aromatic ring is close to the heme iron, in a position appropriate for p-hydroxylation leading to the formation of the product seen only in the presence of the reductase. In the case of codeine, another substrate for cytochrome P450 2D6, the addition of reductase had no effect on the nature of the product formed, the dissociation constant, or the orientation in the binding site. These observations show that NADPH-cytochrome P450 reductase has an allosteric effect on the active site of cytochrome P450 2D6 that affects the binding of some substrates but not others.     

Tissue specific expression of FMR-1 provides evidence for a functional role in fragile X syndrome

Prostaglandin E2 levels in isolated rat islets were increased from 64 +/- 11 pg/30 islets when incubated in medium containing 2 mM glucose to 115 +/- 9 pg/30 islets in medium containing 20 mM glucose. In contrast, glyceraldehyde (10 mM) reduced prostaglandin E2 levels to 29 +/- 6 pg/30 islets. Inhibition of glucose metabolism by mannoheptulose (10 mM) abolished the stimulatory effect of glucose on prostaglandin E2 levels and inhibited glucose-induced insulin release. The cyclooxygenase inhibitor, flurbiprofen (20 microM), did not affect insulin release caused by glucose or glyceraldehyde. In the presence of 1 mg/ml bovine serum albumin, insulin secretion induced by 20 mM glucose (6.9 +/- 1.1% of islet insulin content) was reduced by the lipoxygenase inhibitor BW755 C (20 microM) to 3.1 +/- 0.6%, and by the phospholipase A2 inhibitor, p-bromophenacyl bromide (10 microM), to 2.1 +/- 0.8%. In the absence of bovine serum albumin the inhibitory action of BW755 C and p-bromophenacyl bromide on glucose-induced insulin release was significantly more pronounced. These drugs whether in the presence or absence of bovine serum albumin, did not affect glyceraldehyde-stimulated insulin secretion. Glyceraldehyde (10 mM), potentiated glucose-induced insulin release in the presence of 2-8 mM glucose, but not for 10-20 mM glucose. Although the phospholipase A2 activator, melittin, initiated insulin release in the presence of 2 mM glucose and enhanced 10 mM glyceraldehyde-stimulated insulin secretion it had no effect on 20 mM glucose-induced insulin release. These two stimulatory effects of melittin on insulin release were totally abolished by p-bromophenacyl bromide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mixing and solid-liquid mass-transfer rates in a creusot-loire uddeholm vessel: A water model case study

The process of mixing and solid-liquid mass transfer in a one-fifth scale water model of a 100-ton Creusot-Loire Uddeholm (CLU) converter was investigated. The modified Froude number was used to relate gas flow rates between the model and its protoype. The influences of gas flow rate between 0.010 and 0.018 m³/s and bath height from 0.50 to 0.70 m on mixing time were examined. The results indicated that mixing time decreased with increasing gas flow rate and increased with increasing bath height. The mixing time results were evaluated in terms of specific energy input and the following correlation was proposed for estimating mixing times in the model CLU converter: T _mix=1.08Q ^−1.05 W ^0.35, where Q (m³/s) is the gas flow rate and W (tons) is the model bath weight. Solid-liquid mass-transfer rates from benzoic acid specimens immersed in the gas-agitated liquid phase were assessed by a weight loss measurement technique. The calculated mass-transfer coefficients were highest at the bath surface reaching a value of 6.40 × 10⁻⁵ m/s in the sprout region. Mass-transfer coefficients and turbulence parameters decreased with depth, reaching minimum values at the bottom of the vessel.     

The CADE-14 ATP System Competition

This paper documents the design, competing systems, results, and conclusions of the CADE-14 ATP System Competition (CASC-14).     

Tomographic reconstruction from experimentally obtained cone-beam projections

Direct reconstruction in three dimensions for two-dimensional projection data has been achieved by cone-beam reconstruction techniques. In this paper explicit formulas for a cone-beam convolution and back-projection reconstruction algorithm are given in a form which can be easily coded for a computer. The algorithm is justified by analyzing tomographic reconstructions of a uniformly attenuating sphere from simulated noisy projection data. A particular feature of this algorithm is the use of a one-dimensional rather than two-dimensional convolution function, greatly speeding up the reconstruction. The technique is applicable however large the cone angle of data capture and correctly reduces to the pure fan-beam reconstruction technique in the central section of the cone. The method has been applied to data captured on a cone-beam CT scanner designed for bone mineral densitometry.     

Supporting scenario-based requirements engineering

Scenarios have been advocated as a means of improving requirements engineering yet few methods or tools exist to support scenario based RE. The paper reports a method and software assistant tool for scenario based RE that integrates with use case approaches to object oriented development. The method and operation of the tool are illustrated with a financial system case study. Scenarios are used to represent paths of possible behavior through a use case, and these are investigated to elaborate requirements. The method commences by acquisition and modeling of a use case. The use case is then compared with a library of abstract models that represent different application classes. Each model is associated with a set of generic requirements for its class, hence, by identifying the class(es) to which the use case belongs, generic requirements can be reused. Scenario paths are automatically generated from use cases, then exception types are applied to normal event sequences to suggest possible abnormal events resulting from human error. Generic requirements are also attached to exceptions to suggest possible ways of dealing with human error and other types of system failure. Scenarios are validated by rule based frames which detect problematic event patterns. The tool suggests appropriate generic requirements to deal with the problems encountered. The paper concludes with a review of related work and a discussion of the prospects for scenario based RE methods and tools     

The TPTP Problem Library and Associated Infrastructure

This paper describes the First-Order Form (FOF) and Clause Normal Form (CNF) parts of the TPTP problem library, and the associated infrastructure. TPTP v3.5.0 was the last release containing only FOF and CNF problems, and thus serves as the exemplar. This paper summarizes the history and development of the TPTP, describes the structure and contents of the TPTP, and gives an overview of TPTP related projects and tools.     

High density net shape components by direct laser re-melting of single-phase powders

Direct Metal Laser Re-Melting is a variant of the Selective Laser Sintering process, a Rapid Prototyping (RP) technology. This tool-less manufacturing technology has the potential of producing complex, high quality components from single-phase metal powders in short time scales. This is made possible by the production of consecutive two-dimensional layers. Unfortunately, finished components manufactured by this technique have their integrity and material properties dictated by the porosity within the laser re-melted structure. In order to maintain structural integrity comparable to conventionally produced components, metal components produced by the rapid prototyping method should exhibit a porosity of the order of maximum of 2% with corresponding bulk material properties. To achieve these objectives, process and laser parameters require optimisation for maximum densities to be attained. This paper reports on the development of a scanning strategy that produces stainless steel (316L) laser re-melted components which exhibit porosities of <1%, while maintaining the concept of rapid prototyping.     

Scenario-based assessment of nonfunctional requirements

This paper describes a method and a tool for validating nonfunctional requirements in complex socio-technical systems. The system requirements analyzer (SRA) tool validates system reliability and operational performance requirements using scenario-based testing. Scenarios are transformed into sequences of task steps and the reliability of human agents performing tasks with computerized technology is assessed using Bayesian belief network (BN) models. The tool tests system performance within an envelope of environmental variations and reports the number of tests that pass a benchmark threshold. The tool diagnoses problematic areas in scenarios representing pathways through system models, assists in the identification of their causes, and supports comparison of alternative requirements specifications and system designs. It is suitable for testing socio-technical systems where operational scenarios are sequential and deterministic, in domains where designs are incrementally modified so set up costs of the BNs can be defrayed over multiple tests.     

Microstructural effects on microbial survival: phase-separating dextran solutions

Evolving microstructure in a model dextran solution is shown to exert a major influence on the survival of Escherichia coli K-12 frag 1 and Salmonella typhimurium LT2. The microstructure results from microscopic phase separation, which develops over several hours resulting in hardening of the solution into a glassy state. The microstructure is characterized by an array of physical methods including image analysis, electron spin resonance and bulk rheology, and it is shown that bacterial survival depends on the formation of microscopic. water-rich domains and not primarily on bulk water activity or hardness.