Role of the glutathione S -transferase P1 (GSTP1) electrophile binding site (H-site) in protection from doxorubicin -mediated cytotoxicity

The hypothesis addressed in this project was that novel variants of naturally occurring human glutathione S-transferase P1 (GSTP1) can be created by random mutagenesis of the GSTP1 active site to yield polypeptides with increased enzymatic activity against electrophilic substrates. Specifically, the mutant proteins would metabolize and inactivate selected electrophiles more efficiently than wild-type GSTP1 and confer significant cytoprotection, as measured by reduced apoptosis and increased clonogenic survival. Glutathione S-transferase P1, a major electrophile metabolizing and detoxifying enzyme, is encoded by a polymorphic genetic locus. This locus contains nucleotide transitions in the region encoding the active site of the peptide that yields proteins with significant structural and functional differences. The method of Degenerate Oligonucleotide Mediated Random Mutagenesis (DOMRM) was used to generate cDNAs encoding unique GSTP1 polypeptides with mutations within electrophile binding site (H-site) while leaving the glutathione binding site unaffected. A prokaryotic expression library of the mutant GSTP1 polypeptides was created and screened for increased resistance to cisplatin. This screen resulted in the isolation of 96 clones representing 22 distinct mutant cDNA sequences. To investigate the effects of the changes in the H-site on the biological activity of GSTP1, the cDNA of wild-type GSTP1c and two of the identified mutants were stably transfected into human LNCaP-Pro5 prostate cancer cells that do not endogenously express GSTP1. Wild-type transfectants were resistant to doxorubicin-induced apoptosis and displayed increased clonogenic survival compared to vector controls. However, contrary to the hypothesis, in both assays the mutant transfectants were no more resistant to doxorubicin than the wild-type transfectants. To elucidate the mechanisms underlying GSTP1-mediated survival, an in-vitro assay was developed to determine whether active GSTP1 protein directly metabolizes doxorubicin by conjugation to reduced glutathione (GSH). Although GSH did promote the appearance of a unique doxorubicin conjugate, conjugate formation was not substantially increased by the addition of GSTP1 in a variety of reaction conditions. ^

Sulfuric acid rain effects on crop yield and foliar injury /

Mode of access: Internet.

Effect of different doses of post-emergence-applied iodosulfuron on weed control and grain yield of malt barley (Hordeum distichum L.), under Mediterranean conditions

A study was carried out over a two year period (2009/2010 and 2012/2013) on an experimental farm in the Alentejo region (Beja), in southern Portugal where rainfed malt barley (Hordeum distichum L.) is sown at the end of autumn or beginning of winter (November– December). The aim of this experiment was to study the efficiency of the herbicide iodosulfuron-methyl-sodium to control post-emergence broadleaved weeds in this cereal crop. The malt barley crop was established using no-till farming. This technology provides the necessary machine bearing capacity of the soil to assure the post-emergence application of herbicides at two diferente weed development stages. The herbicide iodosulfuron-methyl-sodium was applied at three doses (5.0, 7.5, and 10.0 g a. i. · ha–1) and at two different broadleaved weed development stages (3 to 4 and 6 to 7 pairs of leaves), that also corresponded to two diferente crop development stages (beginning of tillering and complete tillering). The results indicated that early herbicide application timing provided a significantly higher efficiency for all the applied herbicide doses, but this better weed control was not reflected in a higher crop grain yield. The lack of a higher crop grain yield was probably due to a crop phytotoxicity of the herbicide, when used at an early application timing.

Misunderstanding or Misdirection?: Whistleblower Protection in Queensland

In his report into corruption in Queensland, Fitzgerald listed whistleblower protection as a necessary part of a strong governance regime. "What is required is an accessible, independent body to which disclosures can be made, confidentially (at least in the first instance) and in any event free from fear of reprisals." It was one of the reforms studied by the Electoral and Administrative Review Committee, the report of which resulted in the Whistleblowers Protection Act 1994 (WPA). The need for whistleblower protection was supported by all sides of Parliament. The Premier, Wayne Goss, in his Second Reading Speech on the Public Sector Ethics Bill , said that that Act and the WPA would form a package with the former outlining required behaviour and the WPA encouraging staff to report wrongdoing. The WPA was subsequently passed and has remained virtually unamended for over a decade. Such consistency is either an indication of skilled drafting and effectiveness or the fact that the Act has been neglected. It is the hypothesis of this paper that the latter is the case. This hypothesis will be tested by examining the sincerity and diligence with which the Act has been treated during, and following, its passage.