Domain flexibility in retroviral proteases: structural implications for drug resistant mutations

Rigid body rotation of five domains and movements within their interfacial joints provide a rational context for understanding why HIV protease mutations that arise in drug resistant strains are often spatially removed from the drug or substrate binding sites. Domain motions associated with substrate binding in the retroviral HIV-1 and SIV proteases are identified and characterized. These motions are in addition to closure of the flaps and result from rotations of approximately 6-7 degrees at primarily hydrophobic interfaces. A crystal structure of unliganded SIV protease (incorporating the point mutation Ser 4 His to stabilize the protease against autolysis) was determined to 2.0 A resolution in a new space group, P3221. The structure is in the most "open" conformation of any retroviral protease so far examined, with six residues of the flaps disordered. Comparison of this and unliganded HIV structures, with their respective liganded structures by difference distance matrixes identifies five domains of the protease dimer that move as rigid bodies against one another: one terminal domain encompassing the N- and C-terminal beta sheet of the dimer, two core domains containing the catalytic aspartic acids, and two flap domains. The two core domains rotate toward each other on substrate binding, reshaping the binding pocket. We therefore show that, for enzymes, mutations at interdomain interfaces that favor the unliganded form of the target active site will increase the off-rate of the inhibitor, allowing the substrate greater access for catalysis. This offers a mechanism of resistance to competitive inhibitors, especially when the forward enzymatic reaction rate exceeds the rate of substrate dissociation.     

Lamb wave scattering analysis for reflector characterization

The potential use of guided waves for defect characterization is studied. The influence of defect shape and size on transmitted and reflected fields is considered. Using the hybrid boundary element technique, the reflection and transmission coefficients for selected guided wave modes are numerically calculated and compared to experimental data. Selecting the aspect ratio as a shape parameter for various defects, the transmission and reflection coefficients are measured for certain guided wave modes input to the defect. The influence of defect size is then studied by monitoring the transmission and reflection coefficients for defects of various shapes and depths. The studies presented indicate that defect characterization is possible if a proper mode selection criteria can be established. The suitable features related to transmission and reflection coefficient data can also be used for algorithm development and implementation purposes of defect characterization.     

Degradation of nerve agents by an organophosphate-degrading agent (OpdA)

Enzyme-catalysed degradation of the nerve agents tabun, sarin, ethyl sarin and soman by three variants of an organophosphate-degrading enzyme was studied at low concentrations of nerve agent. The concentration of nerve agent at a given time was determined by its ability to inhibit the enzyme acetylcholinesterase. Experiments were conducted in 96-well microtitre plates. Values of the ratio of k(cat) (turnover number) to K(m) (Michaelis-Menten constant) were calculated. For tabun, this value (for the most effective OpdA variant) exceeded any value published to date for other enzymes. The value was within an order of magnitude for the highest value reported for sarin, but there appears to be no published value for ethyl sarin for comparison. The OpdA enzymes were relatively inefficient in degrading soman.     

Knowing and Not Knowing About Trauma: Implications for Therapy.

Levels of awareness for trauma and their consequences for research, treatment, and prevention within professional psychology and society are considered. When people must endure chronically traumatic environments, it may be adaptive to isolate from awareness information that would produce cognitive dissonance and threaten necessary relationships. Unawareness may also facilitate functioning in environments that invalidate the prevalence and impact of trauma. In addition, characteristics of the posttraumatic environment can promote or impede individuals' awareness of trauma and their psychological functioning. Though often initially adaptive, unawareness for trauma is linked to intergenerational transmission of trauma and its effects and may preclude public and professional attention to trauma treatment and prevention. Understanding the processes through which individuals become unaware or aware of traumatic experience is therefore essential to conducting effective psychotherapy with trauma survivors. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Studies of microhardness and mar resistance using a scanning probe microscope

Scanning probe microscopy (SPM) is in a period of rapid development. It shows great promise for characterizing coating surfaces. This paper describes modification of an SPM so that it can be used to mar the surfaces of coatings under controlled conditions and to characterize the mars. Mar resistance of coatings is analyzed in terms of a ‘three response, two mechanism model.' The three responses (fracture, elastic, and plastic) can be measured quantitatively using the SPM. Of the three responses, only two (fracture and plastic deformation) are marring mechanisms – elastic deformations recover instantaneously. In some cases mars resulting from plastic deformation may recover slowly with time or with immersion in water; this phenomenon is attributed to viscoelastic creep. Microhardness is also measured with the modified SPM. Some thermoset coatings appear to be substantially harder near their surfaces than in the mass of material, and such materials may respond quite differently to stress applied at different levels near the surface. This finding has important implications for all coating properties that are strongly influenced by the surface. A quantity called ‘micro mar resistance' is defined. It may be useful for comparing different coatings under specified conditions of marring. However, there can be no single quantity that expresses ‘mar resistance' of a coating under all conditions.     

Diagnosing Transformers Getting to the Heart of the Matter

From drying your hair in the morning to using your computer at work to watching a DVD on your big screen TV at night, transformers are involved in the many things we do every day. As the name implies, transformers transform electricity from one voltage to another. In the early days of electricity, it was costly to move electricity from Point A to Point B. As a result, people who wanted electricity had to live very close to a power plant. At the 1893 World’s Fair, George Westinghouse and Nikola Tesla introduced alternating current, or AC power, to the world. Up until this time, Thomas Edison’s direct current, or DC power, was the only form of electricity in the United States. Among the many positive attributes of AC power, engineers discovered that if the power could be converted (or transformed) to use higher voltage and lower current, then it would be cost-effective to transport the electricity over longer distances from a power plant to a home or business. This discovery and the invention of the transformer led to the power distribution system as we know it today. Transformers are the backbone of our electrical transmission and distribution system. Of these, high- and medium-voltage transformers are the most complex and the most expensive pieces of equipment in this system. Consequently, it is important that transformers be properly constructed and maintained to ensure a long service life.     

Mechanical Properties and Microstructural Characteristics of Friction Welded Dissimilar Joints of Aluminium Alloys

Joining of similar and dissimilar combinations of aluminium alloys 2024 and 6061 were performed using friction welding technique. Microstructure, hardness and tensile properties of the joints were characterized. Microstructure of the alloy were found to change significantly across the joint such as fully deformed, partially deformed and undeformed regions due to deformation, frictional heat and alloy characteristics. Extensive fine grain size was observed in the fully deformed region and volume fraction of finer grains was higher in the alloy 2024 as compared to alloy 6061. Hardness was lower in the weld interface region of the similar joints of AA2024 and AA6061. The lower hardness in the dissimilar metal joint was observed in the heat affected zone of alloy 6061. The tensile strengths of the similar joints were 80 and 85% of respective base metal of alloys 2024 and 6061. The strength of the dissimilar metal joint was observed to be similar to the base metal strength of 6061 alloy. Tensile fracture occurred in the region of joints where lower hardness was observed. The maximum elongation were obtained in dissimilar joints of alloys and characterized by scanning electron microscope. It revealed deep dimple patterns unlike what was observed in similar joints.     

Biochemical and Structural Characterization of XoxG and XoxJ and Their Roles in Lanthanide-Dependent Methanol Dehydrogenase Activity

Lanthanide (Ln)-dependent methanol dehydrogenases (MDHs) have recently been shown to be widespread in methylotrophic bacteria. Along with the core MDH protein, XoxF, these systems contain two other proteins, XoxG (a c-type cytochrome) and XoxJ (a periplasmic binding protein of unknown function), about which little is known. In this work, we have biochemically and structurally characterized these proteins from the methyltroph Methylobacterium extorquens AM1. In contrast to results obtained in an artificial assay system, assays of XoxFs metallated with La^III, Ce^III, and Nd^III using their physiological electron acceptor, XoxG, display Ln-independent activities, but the K_m for XoxG markedly increases from La to Nd. This result suggests that XoxG′s redox properties are tuned specifically for lighter Lns in XoxF, an interpretation supported by the unusually low reduction potential of XoxG (+172 mV). The X-ray crystal structure of XoxG provides a structural basis for this reduction potential and insight into the XoxG–XoxF interaction. Finally, the X-ray crystal structure of XoxJ reveals a large hydrophobic cleft and suggests a role in the activation of XoxF. These studies enrich our understanding of the underlying chemical principles that enable the activity of XoxF with multiple lanthanides in vitro and in vivo.     

Effect of sugars on the association between cowpea vicilin (7S storage proteins) and fungal cells

Vicilins (7S storage proteins) found in various legume seeds have been previously shown to interfere with the germination of spores or conidia of phytopathogenic fungi and inhibit yeast growth and glucose stimulated acidification of the medium by yeast cells. In the present work vicilins from cowpea (Vigna unguiculata) seeds were added to the growth medium of Saccharomyces cerevisiae cells and Fusarium oxysporum conidia. Helix pomatia lectin, wheat germ agglutinin and Ulex europaeus lectin were used to identify differences in the binding of the vicilins to the surface of cells of S. cerevisiae and F. oxysporum treated with this protein. After the growth period, the material in suspension (yeast cells) was centrifuged and the final pellet was also treated with different sugar (glucose, sucrose, glucosamine, N-acetyl-glucosamine) concentrations and 0.1 M HCl for extraction of vicilins associated to chitinous structures present in yeast cells. Our results showed that vicilin sub-units were present in the different sugar extracts of yeast cells pretreated with the vicilins and these proteins were eluted by 0.5 M solutions of sugars in the following order of efficiency of elution: N-acetyl-glucosamine, sucrose/glucose and glucosamine.