Displacement of a DNA binding protein by Dda helicase

Bacteriophage T4 Dda helicase has recently been shown to be active as a monomer for unwinding of short duplex oligonucleotides and for displacing streptavidin from 3′-biotinylated oligonucleotides. However, its activity for streptavidin displacement and DNA unwinding has been shown to increase as the number of Dda molecules bound to the substrate molecule increases. A substrate was designed to address the ability of Dda to displace DNA binding proteins. A DNA binding site for the Escherichia coli trp repressor was introduced into an oligonucleotide substrate for Dda helicase containing single-stranded overhang. Here we show that a Dda monomer is insufficient to displace the E.coli trp repressor from dsDNA under single turnover conditions, although the substrate is unwound and the repressor displaced when the single-stranded overhang is long enough to accommodate two Dda molecules. The quantity of product formed increases when the substrate is able to accommodate more than two Dda molecules. These results indicate that multiple Dda molecules act to displace DNA binding proteins in a manner that correlates with the DNA unwinding activity and streptavidin displacement activity. We suggest a cooperative inchworm model to describe the activities of Dda helicase

Limitations for change detection in multiple Gabor targets

We investigate the limitations on the ability to detect when a target has changed, using Gabor targets as simple quantifiable stimuli. Using a partial report technique to equalise response variables, we show that the log of the Weber fraction for detecting a spatial frequency change is proportional to the log of the number of targets, with a set-size effect that is greater than that reported for visual search. This is not a simple perceptual limitation, because pre-cueing a single target out of four restores performance to the level found when only one target is present. It is argued that the primary limitation on performance is the division of attention across multiple targets, rather than decay within visual memory. However in a simplified change detection experiment without cueing, where only one target of the set changed, not only was the set size effect still larger, but it was greater at 2000 msec ISI than at 250 msec ISI, indicating a possible memory component. The steepness of the set size effects obtained suggests that even moderate complexity of a stimulus in terms of number of component objects can overload attentional processes, suggesting a possible low-level mechanism for change blindness

Theory of synthetic aperture radar ocean imaging: A MARSEN view

This paper reviews basic synthetic aperture radar (SAR) theory of ocean wave imaging mechanisms, using both known work and recent experimental and theoretical results from the Marine Remote Sensing (MARSEN) Experiment. Several viewpoints that have contributed to the field are drawn together in a general analysis of the backscatter statistics of a moving sea surface. A common focus for different scattering models is provided by the mean image impulse response function, which is shown to be identical to the (spatially varying) frequency variance spectrum of the local complex reflectivity coefficient. From the analysis has emerged a more complete view of the SAR imaging phenomenon than has been previously available. A new, generalized imaging model is proposed

Interactions of ingested food, beverage, and tobacco components involving human cytochrome P4501A2, 2A6, 2E1, and 3A4 enzymes.

Human cytochrome P450 (P450) enzymes are involved in the oxidation of natural products found in foods, beverages, and tobacco products and their catalytic activities can also be modulated by components of the materials. The microsomal activation of aflatoxin B1 to the exo-8,9-epoxide is stimulated by flavone and 7,8-benzoflavone, and attenuated by the flavonoid naringenin, a major component of grapefruit. P4502E1 has been demonstrated to play a potentially major role in the activation of a number of very low-molecular weight cancer suspects, including ethyl carbamate (urethan), which is present in alcoholic beverages and particularly stone brandies. The enzyme (P4502E1) is also known to be inducible by ethanol. Tobacco contains a large number of potential carcinogens. In human liver microsomes a significant role for P4501A2 can be demonstrated in the activation of cigarette smoke condensate. Some of the genotoxicity may be due to arylamines. P4501A2 is also inhibited by components of crude cigarette smoke condensate. The tobacco-specific nitrosamines are activated by a number of P450 enzymes. Of those known to be present in human liver, P4501A2, 2A6, and 2E1 can activate these nitrosamines to genotoxic products

Orientation to pollution prevention for facility design

This material was developed to assist engineers in incorporating pollution prevention into the design of new or modified facilities within the U.S. Department of Energy (DOE). The material demonstrates how the design of a facility can affect the generation of waste throughout a facility`s entire life and it offers guidance on how to prevent the generation of waste during design. Contents include: Orientation to pollution prevention for facility design training course booklet; Pollution prevention design guideline; Orientation to pollution prevention for facility design lesson plan; Training participant survey and pretest; and Training facilitator`s guide and schedule

Road Network Simulation Using FLAME GPU

Demand for high performance road network simulation is increasing due to the need for improved traffic management to cope with the globally increasing number of road vehicles and the poor capacity utilisation of existing infrastructure. This paper demonstrates FLAME GPU as a suitable Agent Based Simulation environment for road network simulations, capable of coping with the increasing demands on road network simulation. Gipps’ car following model is implemented and used to demonstrate the performance of simulation as the problem size is scaled. The performance of message communication techniques has been evaluated to give insight into the impact of runtime generated data structures to improve agent communication performance. A custom visualisation is demonstrated for FLAME GPU simulations and the techniques used are described

Investigation of translocation, DNA unwinding, and protein displacement by NS3h, the helicase domain from the Hepatitis C virus helicase

Helicases are motor proteins that are involved in DNA and RNA metabolism, replication, recombination, transcription and repair. The motors are powered by ATP binding and hydrolysis. Hepatitis C virus encodes a helicase called non-structural protein (NS3). NS3 possesses protease and helicase activities on its N-terminal and C-terminal domains respectively. The helicase domain of NS3 protein is referred as NS3h. In vitro, NS3h catalyzes RNA and DNA unwinding in a 3’ to -5’ direction. The directionality for unwinding is thought to arise in part from the enzyme's ability to translocate along DNA, but translocation has not been shown explicitly. We examined the DNA translocase activity of NS3h by using single-stranded oligonucleotide substrates containing a fluorescent probe on the 5’ end. NS3h can bind to the ssDNA and in the presence of ATP, move towards the 5’-end. When the enzyme encounters the fluorescent probe, a fluorescence change is observed that allows translocation to be characterized. Under conditions that favor binding of one NS3h per DNA substrate (100 nM NS3h, 200 nM oligonucleotide) we find that NS3h translocates on ssDNA at a rate of 46 ± 5 nt s−1 and that it can move for 230 ± 60 nt before dissociating from the DNA. The translocase activity of some helicases is responsible for displacing proteins that are bound to DNA. We studied protein displacement by using a ssDNA oligonucleotide covalently linked to biotin on the 5’-end. Upon addition of streptavidin, a ‘protein-block’ was placed in the pathway of the helicase. Interestingly, NS3h was unable to displace streptavidin from the end of the oligonucleotide, despite its ability to translocate along the DNA. The DNA unwinding activity of NS3h was examined using a 22 bp duplex DNA substrate under conditions that were identical to those used to study translocation. NS3h exhibited little or no DNA unwinding under single cycle conditions, supporting the conclusion that NS3h is a relatively poor helicase in its monomeric form, as has been reported. In summary, NS3h translocates on ssDNA as a monomer, but the translocase activity does not correspond to comparable DNA unwinding activity or protein-displacement activity under identical conditions

The removal of thermally aged films of triacylglycerides by surfactant solutions

Thermal ageing of triacylglycerides (TAG) at high temperatures produces films which resist removal using aqueous surfactant solutions. We used a mass loss method to investigate the removal of thermally aged TAG films from hard surfaces using aqueous solutions of surfactants of different charge types. It was found that cationic surfactants are most effective at high pH, whereas anionics are most effective at low pH and a non-ionic surfactant is most effective at intermediate pH. We showed that the TAG film removal process occurs in several stages. In the first ‘‘lag phase’’ no TAG removal occurs; the surfactant first partitions into the thermally aged film. In the second stage, the TAG film containing surfactant was removed by solubilisation into micelles in the aqueous solution. The effects of pH and surfactant charge on the TAG removal process correlate with the effects of these variables on the extent of surfactant partitioning to the TAG film and on the maximum extent of TAG solubilisation within the micelles. Additionally, we showed how the TAG removal is enhanced by the addition of amphiphilic additives such as alcohols which act as co-surfactants. The study demonstrates that aqueous surfactant solutions provide a viable and more benign alternative to current methods for the removal of thermally aged TAG films