Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications

A set of yeast strains based on Saccharomyces cerevisiae S288C in which commonly used selectable marker genes are deleted by design based on the yeast genome sequence has been constructed and analysed. These strains minimize or eliminate the homology to the corresponding marker genes in commonly used vectors without significantly affecting adjacent gene expression. Because the homology between commonly used auxotrophic marker gene segments and genomic sequences has been largely or completely abolished, these strains will also reduce plasmid integration events which can interfere with a wide variety of molecular genetic applications. We also report the construction of new members of the pRS400 series of vectors, containing the kanMX, ADE2 and MET15 genes.     

Conduction block in vasculitic neuropathy

Vasculitis involving peripheral nerves usually presents as an acute asymmetrical axonal neuropathy. We report a 67-year-old man with a symmetrical subacute neuropathy in which nerve conduction studies showed prominent conduction block, a finding indicative of demyelination. Sural nerve biopsy showed a vasculitic neuropathy with invasion of blood vessel walls by inflammatory cells and a mixture of nerve fiber loss and demyelination. The demyelination in this case was presumably a consequence of subinfarctive nerve ischemia.     

Hydrophilic peptides derived from the transframe region of Gag-Pol inhibit the HIV-1 protease

The HIV-1 transframe region (TFR) is between the structural and functional domains of the Gag-Pol polyprotein, flanked by the nucleocapsid and the protease domains at its N and C termini, respectively. Transframe octapeptide (TFP) Phe-Leu-Arg-Glu-Asp-Leu-Ala-Phe, the N terminus of TFR, and its analogues are competitive inhibitors of the action of the mature HIV-1 protease. The smallest, most potent analogues are tripeptides: Glu-Asp-Leu and Glu-Asp-Phe with Ki values of approximately 50 and approximately 20 microM, respectively. Substitution of the acidic amino acids in the TFP by neutral amino acids and d or retro-d configurations of Glu-Asp-Leu results in an >40-fold increase in Ki. Protease inhibition by Glu-Asp-Leu is dependent on a protonated form of a group with a pKa of 3.8; unlike other inhibitors of HIV-1 protease which are highly hydrophobic, Glu-Asp-Leu is extremely soluble in water, and its binding affinity decreases with increasing NaCl concentration. However, Glu-Asp-Leu is a poor inhibitor (Ki approximately 7.5 mM) of the mammalian aspartic acid protease pepsin. X-ray crystallographic studies at pH 4.2 show that the interactions of Glu at P2 and Leu at P1 of Glu-Asp-Leu with residues of the active site of HIV-1 protease are similar to those of other product-enzyme complexes. It was not feasible to understand the interaction of intact TFP with HIV-1 protease under conditions of crystal growth due to its hydrolysis giving rise to two products. The sequence-specific, selective inhibition of the HIV-1 protease by the viral TFP suggests a role for TFP in regulating protease function during HIV-1 replication.     

Caring for dying children: nurses' experiences

Interviews with 25 nurses in this grounded theory study show that when nurses recognized that a child's death was inevitable, they struggled with both grief distress and moral distress. Their distress occurred within the context of the nurse-patient relationship. Nurses employed a range of strategies to manage their distress. Several conditions facilitated or constrained nurses' strategies, and resulted in far-reaching implications both professionally and personally.     

Effect of precipitate type and morphology on high-temperature creep and internal stress in Al-Li based alloys

The tensile creep of a series of aluminium-lithium-based alloys, two binary alloys containing precipitate, and the 2090 alloy containing and T₁ precipitate, has been studied over a range of stresses at 150°C. In some cases the internal stress developed during creep has been determined using the strain transient dip test. The results have been compared with similar data previously obtained for the 8090 alloy containing and S precipitates. The solid solution alloy and the binary alloy containing shearable particles exhibited normal Class II behaviour, with the development of sub-grains and a stress dependence of the creep rate given by a single stress exponent,n, between 4 and 5 at all applied stresses. The alloys containing particles not easily sheared by dislocations, coarse , S and T₁, exhibited similar stress dependencies of the creep rate at low stresses but exhibited large values ofn, between 18 and 35 at high stresses. The internal stress, _i, in these alloys was found to be approximately constant at high stresses possibly due to partial shearing of the coarse , T₁, and the S on sub-boundaries. The stress dependence of the minimum creep rate, , could be represented at all applied stresses, _a, by , where (_a–_i) is the effective stress driving dislocations during creep, andn is a single stress exponent of between 5 and 6 for all applied stresses. The internal stress, which increases with applied stress, at least at a low applied stress, arises from inhomogeneity of plastic deformation, due to hard sub-boundaries or hard particles which are Orowan looped. These two types of contribution to the internal stress are of similar magnitude in the alloys containing coarse and T₁ but the majority of the internal stress in the 8090 alloy may arise as a result of the hardening of sub-boundaries by the S precipitate.     

Annexin 1 Is a Component of eATP-Induced Cytosolic Calcium Elevation in Arabidopsis thaliana Roots

Extracellular ATP (eATP) has long been established in animals as an important signalling molecule but this is less understood in plants. The identification of Arabidopsis thaliana DORN1 (Does Not Respond to Nucleotides) as the first plant eATP receptor has shown that it is fundamental to the elevation of cytosolic free Ca²⁺ ([Ca²⁺]_cyt) as a possible second messenger. eATP causes other downstream responses such as increase in reactive oxygen species (ROS) and nitric oxide, plus changes in gene expression. The plasma membrane Ca²⁺ influx channels involved in eATP-induced [Ca²⁺]_cyt increase remain unknown at the genetic level. Arabidopsis thaliana Annexin 1 has been found to mediate ROS-activated Ca²⁺ influx in root epidermis, consistent with its operating as a transport pathway. In this study, the loss of function Annexin 1 mutant was found to have impaired [Ca²⁺]_cyt elevation in roots in response to eATP or eADP. Additionally, this annexin was implicated in modulating eATP-induced intracellular ROS accumulation in roots as well as expression of eATP-responsive genes.     

On The Measurement of Temperature in Material Removal Processes

Because it is key to understanding the performance of material removal processes and resultant workpiece quality, the measurement of temperature during material removal is done extensively. We review several widely used temperature measurement methods and show how they can be applied to temperature monitoring during material removal. Since there is little documentation on measurement uncertainties as relates to material removal, this paper outlines the physics of each method, detailing the sources and evaluation of uncertainty. Finally, using criteria critical in measuring material removal, methods are compared, and the results presented in guide-format for participants in this field of work.     

The effect of protein binding on ibuprofen adsorption to activated carbons

The potential of a range of polymer-based activated carbons to remove a non-steroidal anti-inflammatory drug ibuprofen from protein containing and protein-free solutions was studied. The adsorption capacity of five activated carbons produced from phenol-formaldehyde resin precursors (MAST Carbon Ltd) was compared to a cellulose coated activated carbon Norit RBX used in a commercial Adsorba^®300C haemoperfusion column (Gambro).Ibuprofen adsorption was studied in batch experiments over 24 h at pH=7.6 and 25 °C and adsorption equilibrium data were correlated with the Langmuir and Freundlich equations.Results showed that uncoated mesoporous MAST carbons can remove ibuprofen from model solutions both in the presence and absence of serum albumin. Ibuprofen is a highly protein-bound drug and the presence of serum albumin significantly affects the adsorption of ibuprofen by cellulose-coated microporous carbon used in Adsorba^®300C column.     

Inheritance Of Resistance To Mammalian Herbivores and Of Plant Defensive Chemistry In A <Emphasis Type="Italic">Eucalyptus</Emphasis> Species

Hybridization in plants provides an opportunity to investigate the patterns of inheritance of hybrid resistance to herbivores, and of the plant mechanisms conferring this resistance such as plant secondary metabolites. We investigated how inter-race differences in resistance of Eucalyptus globulus to a generalist mammalian herbivore, Trichosurus vulpecula, are inherited in their F₁ hybrids. We assessed browsing damage of three-year-old trees in a common environment field trial on four hybrid types of known progeny. The progency were artificial intra-race crosses and reciprocal inter-race F₁ hybrids of two geographically distinct populations (races) of E. globulus; north-eastern Tasmania and south-eastern Tasmania. Populations of trees from north-eastern Tasmania are relatively susceptible to browsing by T. vulpecula, while populations from south-eastern Tasmania are more resistant. We assessed the preferences of these trees in a series of paired feeding trials with captive animals to test the field trial results and also investigated the patterns of inheritance of plant secondary metabolites. Our results demonstrated that the phenotypic expression of resistance of the inter-race F₁ hybrids supported the additive pattern of inheritance, as these hybrids were intermediate in resistance compared to the pure parental hybrids. The expression of plant secondary metabolites in the F₁ hybrids varied among groups of individual compounds. The most common pattern supported was dominance towards one of the parental types. Together, condensed tannins and essential oils appeared to explain the observed patterns of resistance among the four hybrid types. While both chemical groups were inherited in a dominant manner in the inter-race F₁ hybrids, the direction of dominance was opposite. Their combined concentration, however, was inherited in an additive manner, consistent with the phenotypic differences in browsing.