The isoflavonoids genistein and quercetin activate different stress signaling pathways as shown by analysis of site-specific phosphorylation of ATM, p53 and histone H2AX

The ataxia-telangiectasia mutated (ATM) protein kinase is activated in response to ionizing radiation (IR) and activates downstream DNA-damage signaling pathways. Although the role of ATM in the cellular response to ionizing radiation has been well characterized, its role in response to other DNA-damaging agents is less well defined. We previously showed that genistein, a naturally occurring isoflavonoid, induced increased ATM protein kinase activity, ATM-dependent phosphorylation of p53 on serine 15 and activation of the DNA-binding properties of p53. Here. we show that genistein also induces phosphorylation of p53 at serines 6, 9, 20,46, and 392, and that genistein-induced accumulation and phosphorylation of p53 is reduced in two ATM-deficient human cell lines. Also, we show that genistein induces phosphorylation of ATM on serine 1981 and phosphorylation of histone H2AX on serine 139. The related bioflavonoids, daidzein and biochanin A, did not induce either phosphorylation of p53 or ATM at these sites. Like genistein, quercetin induced phosphorylation of ATM on serine 198 1, and ATM-dependent phosphorylation of histone H2AX on serine 139; however, p53 accumulation and phosphorylation on serines 6, 9, 15, 20, 46, and 392 occurred in ATM-deficient cells, indicating that ATM is not required for quercetin-induced phosphorylation of p53. Our data suggest that genistein and quercetin induce different DNA-damage induced signaling pathways that, in the case of genistein, are highly ATM-dependent but, in the case of quercetin, may be ATM-dependent only for some downstream targets. (C) 2003 Elsevier B.V. All rights reserved.

Site-specific labelling of proteins with a rigid lanthanide-binding tag

This paper describes a generic method for the site-specific attachment of lathanide complexes to proteins through a disulfide bond. The method is demonstrated by the attachment of a lanthanide-binding peptide tag to the single cysteine residue present in the N-terminal DNA-binding domain of the Echerichia coli arginine repressor. Complexes with Y3+, Tb3+, Dy3+, Ho3+, Er3+, Tm3+ and Yb3+ ions were formed and analysed by NMR spectroscopy. Large pseudocontact shifts and residual dipolar couplings were induced by the lanthanide-binding tag in the protein NMR spectrum, a result indicating that the tag was rigidly attached to the protein. The axial components of the magnetic susceptibility anisostropy tensors determined for the different lanthanide ions were similarly but not identically oriented. A single tag with a single protein attachment site can provide different pseudocontact shifts from different magnetic susceptibility tensors and thus provide valuable nondegenerate long-range structure information in the determination of 3D protein structures by NMR spectroscopy.

Effect of site-specific mutations in different phosphotransfer domains of the chemosensory protein ChpA on Pseudomonas aeruginosa motility

The virulence of Pseudomonas aeruginosa and other surface pathogens involves the coordinate expression of a wide range of virulence determinants, including type IV pili. These surface filaments are important for the colonization of host epithelial tissues and mediate bacterial attachment to, and translocation across, surfaces by a process known as twitching motility. This process is controlled in part by a complex signal transduction system whose central component, ChpA, possesses nine potential sites of phosphorylation, including six histidine-containing phosphotransfer (HPt) domains, one serine-containing phosphotransfer domain, one threonine-containing phosphotransfer domain, and one CheY-like receiver domain. Here, using site-directed mutagenesis, we show that normal twitching motility is entirely dependent on the CheY-like receiver domain and partially dependent on two of the HPt domains. Moreover, under different assay conditions, point mutations in several of the phosphotransfer domains of ChpA give rise to unusual "swarming" phenotypes, possibly reflecting more subtle perturbations in the control of P. aeruginosa motility that are not evident from the conventional twitching stab assay. Together, these results suggest that ChpA plays a central role in the complex regulation of type IV pilus-mediated motility in P. aeruginosa

Incorporating a TEV cleavage site reduces the solubility of nine recombinant mouse proteins

Failure to express soluble proteins in bacteria is mainly attributed to the properties of the target protein itself, as well as the choice of the vector, the purification tag and the linker between the tag and protein, and codon usage. The expression of proteins with fusion tags to facilitate subsequent purification steps is a widely used procedure in the production of recombinant proteins. However, the additional residues can affect the properties of the protein; therefore, it is often desirable to remove the tag after purification. This is usually done by engineering a cleavage site between the tag and the encoded protein that is recognised by a site-specific protease, such as the one from tobacco etch virus (TEV). In this study, we investigated the effect of four different tags on the bacterial expression and solubility of nine mouse proteins. Two of the four engineered constructs contained hexahistidine tags with either a long or short linker. The other two constructs contained a TEV cleavage site engineered into the linker region. Our data show that inclusion of the TEV recognition site directly downstream of the recombination site of the Invitrogen Gateway vector resulted, in a loss of solubility of the nine mouse proteins. Our work suggests that one needs to be very careful when making modifications to expression vectors and combining different affinity and fusion tags and cleavage sites: (c) 2006 Elsevier Inc. All rights reserved.

STAR: predicting recombination sites from amino acid sequence

Background: Designing novel proteins with site-directed recombination has enormous prospects. By locating effective recombination sites for swapping sequence parts, the probability that hybrid sequences have the desired properties is increased dramatically. The prohibitive requirements for applying current tools led us to investigate machine learning to assist in finding useful recombination sites from amino acid sequence alone. Results: We present STAR, Site Targeted Amino acid Recombination predictor, which produces a score indicating the structural disruption caused by recombination, for each position in an amino acid sequence. Example predictions contrasted with those of alternative tools, illustrate STAR'S utility to assist in determining useful recombination sites. Overall, the correlation coefficient between the output of the experimentally validated protein design algorithm SCHEMA and the prediction of STAR is very high (0.89). Conclusion: STAR allows the user to explore useful recombination sites in amino acid sequences with unknown structure and unknown evolutionary origin. The predictor service is available from http://pprowler.itee.uq.edu.au/star.

Reactions catalyzed by bacterial cytochromes P450

The cytochromes P450 are a large family of oxidative haemoproteins that are responsible for a wide variety of oxidative transformations in a variety of organisms. This review focuses upon the reactions catalyzed specifically by bacterial enzymes, which includes aliphatic hydroxylation, alkene epoxidation, aromatic hydroxylation, oxidative phenolic coupling, heteroatom oxidation and dealkylation, and multiple oxidations including C-C bond cleavage. The potential for the practical application of the oxidizing power of these enzymes is briefly discussed.

Transmission electron microscopy sample preparation technique for sintered alloys

Powder metallurgy alloys are typically inhomogeneous with a significant amount of porosity. This complicates conventional transmission electron microscopy sample preparation. However, the use of focused ion beam milling allows site specific transmission electron microscopy samples to be prepared in a short amount of time. This paper presents a method that can be used to produce transmission electron microscopy samples from an Al-Cu-Mg PM alloy. (C) 2003 IoM Communications Ltd. Published by Maney for the Institute of Materials, Minerals and Mining.

Mapping and characterising subtropical estuarine landscapes using aerial photography and GIS for potential application in wildlife conservation and management

Coarse-resolution thematic maps derived from remotely sensed data and implemented in GIS play an important role in coastal and marine conservation, research and management. Here, we describe an approach for fine-resolution mapping of land-cover types using aerial photography and ancillary GIs and ground data in a large (100 x 35 km) subtropical estuarine system (Moreton Bay, Queensland, Australia). We have developed and implemented a classification scheme representing 24 coastal (subtidal, intertidal. mangrove, supratidal and terrestrial) cover types relevant to the ecology of estuarine animals, nekton and shorebirds. The accuracy of classifications of the intertidal and subtidal cover types, as indicated by the agreement between the mapped (predicted) and reference (ground) data, was 77-88%, depending on the zone and level of generalization required. The variability and spatial distribution of habitat mosaics (landscape types) across the mapped environment were assessed using K-means clustering and validated with Classification and Regression Tree models. Seven broad landscape types could be distinguished and ways of incorporating the information on landscape composition into site-specific conservation and field research are discussed. This research illustrates the importance and potential applications of fine-resolution mapping for conservation and management of estuarine habitats and their terrestrial and aquatic wildlife. (c) 2005 Elsevier Ltd. All rights reserved.

Role of smoking in global and regional cancer epidemiology: current patterns and data needs

Although smoking is widely recognized as a major cause of cancer, there is little information on how it contributes to the global and regional burden of cancers in combination with other risk factors that affect background cancer mortality patterns. We used data from the American Cancer Society's Cancer Prevention Study II (CPS-II) and the WHO and IARC cancer mortality databases to estimate deaths from 8 clusters of site-specific cancers caused by smoking, for 14 epidemiologic subregions of the world, by age and sex. We used lung cancer mortality as an indirect marker for accumulated smoking hazard. CPS-II hazards were adjusted for important covariates. In the year 2000, an estimated 1.42 (95% CI 1.27-1.57) million cancer deaths in the world, 21% of total global cancer deaths, were caused by smoking. Of these, 1.18 million deaths were among men and 0.24 million among women; 625,000 (95% CI 485,000-749,000) smoking-caused cancer deaths occurred in the developing world and 794,000 (95% CI 749,000-840,000) in industrialized regions. Lung cancer accounted for 60% of smoking-attributable cancer mortality, followed by cancers of the upper aerodigestive tract (20%). Based on available data, more than one in every 5 cancer deaths in the world in the year 2000 were caused by smoking, making it possibly the single largest preventable cause of cancer mortality. There was significant variability across regions in the role of smoking as a cause of the different site-specific cancers. This variability illustrates the importance of coupling research and surveillance of smoking with that for other risk factors for more effective cancer prevention. (C) 2005 Wiley-Liss, Inc.

Sustainability metrics for coal power generation in Australia

The basis of this work was to investigate the relative environmental impacts of various power generators knowing that all plants are located in totally different environments and that different receptors will experience different impacts. Based on IChemE sustainability metrics paradigm, we calculated potential environmental indicators (P-EI) that represent the environmental burden of masses of potential pollutants discharged into different receiving media. However, a P-EI may not be of significance, as it may not be expressed at all in different conditions, so to try and include some receiver significance we developed a methodology to take into account some specific environmental indicators (S-EI) that refer to the environmental attributes of a specific site. In this context, we acquired site specific environmental data related to the airsheds and water catchment areas in different locations for a limited number of environmental indicators such as human health (carcinogenic) effects, atmospheric acidification, photochemical (ozone) smog and eutrophication. The S-EI results from this particular analysis show that atmospheric acidification has highest impact value while health risks due to fly ash emissions are considered not to be as significant. This is due to the fact that many coal power plants in Australia are located in low population density air sheds. The contribution of coal power plants to photochemical (ozone) smog and eutrophication were not significant. In this study, we have considered emission related data trends to reflect technology performance (e.g., P-EI indicators) while a real sustainability metric can be associated only with the specific environmental conditions of the relevant sites (e.g., S-EI indicators).

The Pedagogical Practice of Locative Experience

We work collectively with varied locative-type projects and look to integrate our students into contemporary experience design culture. Students experience the ‘how and what’ of locative by becoming participant users, being exposed to contemporary works, and placing themselves in the role of the designer producing their own located works.

In vivo demonstration of load-induced fluid flow in the rat tibia and its potential implications for processes associated with functional adaptation

Load-induced extravascular fluid flow has been postulated to play a role in mechanotransduction of physiological loads at the cellular level. Furthermore, the displaced fluid serves as a carrier for metabolites, nutrients, mineral precursors and osteotropic agents important for cellular activity. We hypothesise that load-induced fluid flow enhances the transport of these key substances, thus helping to regulate cellular activity associated with processes of functional adaptation and remodelling. To test this hypothesis, molecular tracer methods developed previously by our group were applied in vivo to observe and quantify the effects of load-induced fluid flow under four-point-bending loads. Preterminal tracer transport studies were carried out on 24 skeletally mature Sprague Dawley rats. Mechanical loading enhanced the transport of both small- and larger-molecular-mass tracers within the bony tissue of the tibial mid-diaphysis. Mechanical loading showed a highly significant effect on the number of periosteocytic spaces exhibiting tracer within the cross section of each bone. For all loading rates studied, the concentration of Procion Red tracer was consistently higher in the tibia subjected to pure bending loads than in the unloaded, contralateral tibia, Furthermore, the enhancement of transport was highly site-specific. In bones subjected to pure bending loads, a greater number of periosteocytic spaces exhibited the presence of tracer in the tension band of the cross section than in the compression band; this may reflect the higher strains induced in the tension band compared with the compression band within the mid-diaphysis of the rat tibia. Regardless of loading mode, the mean difference between the loaded side and the unloaded contralateral control side decreased with increasing loading frequency. Whether this reflects the length of exposure to the tracer or specific frequency effects cannot be determined by this set of experiments. These in vivo experimental results corroborate those of previous ex vivo and in vitro studies, Strain-related differences in tracer distribution provide support for the hypothesis that load-induced fluid flow plays a regulatory role in processes associated with functional adaptation.

Sulfadoxine resistance in Plasmodium vivax is associated with a specific amino acid in dihydropteroate synthase at the putative sulfadoxine-binding site

Sulfadoxine is predominantly used in combination with pyrimethamine, commonly known as Fansidar, for the treatment of Plasmodium falciparum. This combination is usually less effective against Plasmodium vivax, probably due to the innate refractoriness of parasites to the sulfadoxine component. To investigate this mechanism of resistance by P. vivax to sulfadoxine, we cloned and sequenced the P. vivax dhps (pvdhps) gene. The protein sequence was determined, and three-dimensional homology models of dihydropteroate synthase (DHPS) from P. vivax as well as P. falciparum were created. The docking of sulfadoxine to the two DHPS models allowed us to compare contact residues in the putative sulfadoxine-binding site in both species. The predicted sulfadoxine-binding sites between the species differ by one residue, V585 in P. vivax, equivalent to A613 in P. falciparum. V585 in P. vivax is predicted by energy minimization to cause a reduction in binding of sulfadoxine to DHPS in P. vivax compared to P. falciparum. Sequencing dhps genes from a limited set of geographically different P. vivax isolates revealed that V585 was present in all of the samples, suggesting that V585 may be responsible for innate resistance of P. vivax to sulfadoxine. Additionally, amino acid mutations were observed in some P. vivax isolates in positions known to cause resistance in P. falciparum, suggesting that, as in P. falciparum, these mutations are responsible for acquired increases in resistance of P. vivax to sulfadoxine.