Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency

Because of its stringent sequence specificity, the catalyticdomain of the nuclear inclusion protease from tobacco etch virus(TEV) is a useful reagent for cleaving genetically engineeredfusion proteins. However, a serious drawback of TEV proteaseis that it readily cleaves itself at a specific site to generatea truncated enzyme with greatly diminished activity. The rateof autoinactivation is proportional to the concentration ofTEV protease, implying a bimolecular reaction mechanism. Yet,a catalytically active protease was unable to convert a catalyticallyinactive protease into the truncated form. Adding increasingconcentrations of the catalytically inactive protease to a fixedamount of the wild-type enzyme accelerated its rate of autoinactivation.Taken together, these results suggest that autoinactivationof TEV protease may be an intramolecular reaction that is facilitatedby an allosteric interaction between protease molecules. Inan effort to create a more stable protease, we made amino acidsubstitutions in the P2 and P1' positions of the internal cleavagesite and assessed their impact on the enzyme's stability andcatalytic activity. One of the P1' mutants, S219V, was not onlyfar more stable than the wild-type protease (~100-fold), butalso a more efficient catalyst.     

Low temperature fatigue crack propagation in neutron-irradiated type 316 steel and weld metal

The fast cycling fatigue crack propagation characteristics of type 316 steel and weld metal have been investigated at 380°C after irradiation to 1.72?1.92 × 10²⁰ n/cm²($\text{E > 1}$ MeV) and 2.03 × 10²¹ n/cm² ($\text{E > 1}$ MeV)at the same temperature. With mill-annealed type 316 steel, modest decreases in the rates of crack propagation were observed for both dose levels considered, whereas for cold-worked type 316 steel irradiation to 2.03 × 10²¹ n/cm² ($\text{E > 1}$ MeV) caused increases in the rate of crack propagation. For type 316 weld metal, increases in the rate of crack propagation were observed for both dose levels considered.The diverse influences of irradiation upon fatigue crack propagation in these materials are explained by considering a simple continuum mechanics model of crack propagation, together with the results of control tensile experiments made on similarly irradiated materials.     

Cleavage of poly(A)-binding protein by enterovirus proteases concurrent with inhibition of translation in vitro

Many enteroviruses, members of the family Picornaviridae, cause a rapid and drastic inhibition of host cell protein synthesis during infection, a process referred to as host cell shutoff. Poliovirus, one of the best-studied enteroviruses, causes marked inhibition of host cell translation while preferentially allowing translation of its own genomic mRNA. An abundance of experimental evidence has accumulated to indicate that cleavage of an essential translation initiation factor, eIF4G, during infection is responsible at least in part for this shutoff. However, evidence from inhibitors of viral replication suggests that an additional event is necessary for the complete translational shutoff observed during productive infection. This report examines the effect of poliovirus infection on a recently characterized 3' end translational stimulatory protein, poly(A)-binding protein (PABP). PABP is involved in stimulating translation initiation in lower eukaryotes by its interaction with the poly(A) tail on mRNAs and has been proposed to facilitate 5'-end-3'-end interactions in the context of the closed-loop translational model. Here, we show that PABP is specifically degraded during poliovirus infection and that it is cleaved in vitro by both poliovirus 2A and 3C proteases and coxsackievirus B3 2A protease. Further, PABP cleavage by 2A protease is accompanied by concurrent loss of translational activity in an in vitro-translation assay. Similar loss of translational activity also occurs simultaneously with partial 3C protease-mediated cleavage of PABP in translation assays. Further, PABP is not degraded during infections in the presence of guanidine-HCl, which blocks the complete development of host translation shutoff. These results provide preliminary evidence that cleavage of PABP may contribute to inhibition of host translation in infected HeLa cells, and they are consistent with the hypothesis that PABP plays a role in facilitating translation initiation in higher eukaryotes.     

Light flicker test in the United States

Home appliances, like washers, dryers, refrigerators, etc., can cause voltage fluctuations and light flicker. A standardized flicker test has been established in Europe (IEC 1000-3-3). However, the European test is designed for 230-V 50-Hz power and needs to be modified for a 115-V 60-Hz supply. In this paper, the reference impedance and the limits of flicker tests for 115 V/60 Hz systems are proposed. A high-performance power analysis system, the Xitron 2803AH power analyzer, was used to study the voltage fluctuations and flicker caused by some washing machines. The analysis and test results provided in this paper can serve as a proposal for establishing a US light flicker testing standard for 115 V/60 Hz     

Fast diffusers in a thermal gradient (solder ball)

The behaviour of Ni in Sn under a thermal gradient driving forces was calculated considering boundary conditions typical of lead-free solder applications. The steady state concentration profiles for a fast diffuser were determined and the consequences for reliability discussed.     

Probabilistic modelling, inference and learning using logical theories

This paper provides a study of probabilistic modelling, inference and learning in a logic-based setting. We show how probability densities, being functions, can be represented and reasoned with naturally and directly in higher-order logic, an expressive formalism not unlike the (informal) everyday language of mathematics. We give efficient inference algorithms and illustrate the general approach with a diverse collection of applications. Some learning issues are also considered.     

<Emphasis Type="Italic">Corymbia</Emphasis> Species and Hybrids: Chemical and Physical Foliar Attributes and Implications for Herbivory

Hybridization is an important biological phenomenon that can be used to understand the evolutionary process of speciation of plants and their associated pests and diseases. Interactions between hybrid plants and the herbivores of the parental taxa may be used to elucidate the various cues being used by the pests for host location or other processes. The chemical composition of plants, and their physical foliar attributes, including leaf thickness, trichome density, moisture content and specific leaf weight were compared between allopatric pure and commercial hybrid species of Corymbia, an important subtropical hardwood taxon. The leaf-eating beetle Paropsis atomaria, to which the pure taxa represented host (C. citriodora subsp. variegata) and non-host (C. torelliana) plants, was used to examine patterns of herbivory in relation to these traits. Hybrid physical foliar traits, chemical profiles, and field and laboratory beetle feeding preference, while showing some variability, were generally intermediate to those exhibited by parent taxa, thus suggesting an additive inheritance pattern. The hybrid susceptibility hypothesis was not supported by our field or laboratory studies, and there was no strong relationship between adult preference and larval performance. The most-preferred adult host was the sympatric taxon, although this species supported the lowest larval survival, while the hybrid produced significantly smaller pupae than the pure species. The results are discussed in relation to plant chemistry and physical characteristics. The findings suggest a chemical basis for host selection behavior and indicate that it may be possible to select for resistance to this insect pest in these commercially important hardwood trees.     

The precipitation of the Q phase in an AA6111 Alloy

The precipitation behavior of the quaternary Q phase in an Al 6111 alloy has been studied by analytical electron microscopy. The transformation strain associated with the Q phase has been determined from high resolution electron microscopy and electron diffraction. The habit plane of the Q laths is shown to be fully coherent with the Al matrix. The transformation strain is used to explain the pattern of heterogeneous precipitation of the Q phase at dislocations and grain boundaries. The crystal structure and composition of the Q phase, whether it forms in the matrix or at grain boundaries, appears similar to that formed directly from the melt in a quaternary Al-Cu-Mg-Si alloy.     

Triggering steam explosions of single drops of a molten ferrosilicon alloy with a simple encapsulated mechanical impactor

We have developed a simple and inexpensive encapsulated mechanical impactor that can be operated under water. The unit reproducibly generates upward-directed pressure transients in the water with peaks of approximately 0.13 MPa measured 100 mm above its surface. These pulses have been used to trigger steam explosions of drops of a molten ferrosilicon alloy when they are either just above the unit in free fall through the water or shortly after they land on its surface. The impactor is study, reliable, safe, and environmentally neutral and resets for reuse within seconds. Compared to other triggering techniques, the impactor does not generate light, bubbles, or strong water motion that obscure or appreciably affect the initiation or early stages of the steam explosions. This permits good imaging and measurements at essentially all times during the triggering and subsequent development of the explosions.     

The Management of Knowledge during Design and Implementation of Process Technologies

Knowledge and learning play a major part in the successful implementation of process technologies. We suggest that managers of implementation projects can improve the initial performance levels of the process in which a new technology resides by ensuring that useful knowledge, present in the organization at the start of the implementation project, is employed to greatest effect during the design of the modified process and that new learning is not lost. This does not necessarily require major investment in computerized systems – merely exploring what potentially useful knowledge is already available within the organization, or can readily be obtained from external contact, may improve the efficiency of technology implementation. Use of available knowledge can also help to speed up the implementation process by reducing the number of, and time required for, the adaptations necessary to get the process to the desired performance level.