NEIL1 excises 3′ end proximal oxidative DNA lesions resistant to cleavage by NTH1 and OGG1

Base excision repair is the major pathway for the repair of oxidative DNA damage in human cells that is initiated by a damage-specific DNA glycosylase. In human cells, the major DNA glycosylases for the excision of oxidative base damage are OGG1 and NTH1 that excise 8-oxoguanine and oxidative pyrimidines, respectively. We find that both enzymes have limited activity on DNA lesions located in the vicinity of the 3′ end of a DNA single-strand break, suggesting that other enzymes are involved in the processing of such lesions. In this study, we identify and characterize NEIL1 as a major DNA glycosylase that excises oxidative base damage located in close proximity to the 3′ end of a DNA single-strand break

Structure of the uncomplexed DNA repair enzyme endonuclease VIII indicates significant interdomain flexibility

Escherichia coli endonuclease VIII (Nei) excises oxidized pyrimidines from DNA. It shares significant sequence homology and similar mechanism with Fpg, a bacterial 8-oxoguanine glycosylase. The structure of a covalent Nei–DNA complex has been recently determined, revealing critical amino acid residues which are important for DNA binding and catalysis. Several Fpg structures have also been reported; however, analysis of structural dynamics of Fpg/Nei family proteins has been hindered by the lack of structures of uncomplexed and DNA-bound enzymes from the same source. We report a 2.8 Å resolution structure of free wild-type Nei and two structures of its inactive mutants, Nei-E2A (2.3 Å) and Nei-R252A (2.05 Å). All three structures are virtually identical, demonstrating that the mutations did not affect the overall conformation of the protein in its free state. The structures show a significant conformational change compared with the Nei structure in its complex with DNA, reflecting a ∼50° rotation of the two main domains of the enzyme. Such interdomain flexibility has not been reported previously for any DNA glycosylase and may present the first evidence for a global DNA-induced conformational change in this class of enzymes. Several local but functionally relevant structural changes are also evident in other parts of the enzyme

CLC-2 single nucleotide polymorphisms (SNPs) as potential modifiers of cystic fibrosis disease severity

BACKGROUND: Cystic fibrosis (CF) lung disease manifest by impaired chloride secretion leads to eventual respiratory failure. Candidate genes that may modify CF lung disease severity include alternative chloride channels. The objectives of this study are to identify single nucleotide polymorphisms (SNPs) in the airway epithelial chloride channel, CLC-2, and correlate these polymorphisms with CF lung disease. METHODS: The CLC-2 promoter, intron 1 and exon 20 were examined for SNPs in adult CF dF508/dF508 homozygotes with mild and severe lung disease (forced expiratory volume at one second (FEV1) > 70% and < 40%). RESULTS: PCR amplification of genomic CLC-2 and sequence analysis revealed 1 polymorphism in the hClC -2 promoter, 4 in intron 1, and none in exon 20. Fisher's analysis within this data set, did not demonstrate a significant relationship between the severity of lung disease and SNPs in the CLC-2 gene. CONCLUSIONS: CLC-2 is not a key modifier gene of CF lung phenotype. Further studies evaluating other phenotypes associated with CF may be useful in the future to assess the ability of CLC-2 to modify CF disease severity

The global biopharma industry and the rise of Indian drug multinationals: implications for Australian generics policy

This article provides a synopsis of the new dynamics of the global biopharma industry. The emergence of global generics companies with capabilities approximating those of 'big pharma' has accelerated the blurring of boundaries between the innovator and generics sectors. Biotechnology-based products form a large and growing segment of prescription drug markets and regulatory pathways for biogenerics are imminent. Indian biopharma multinationals with large-scale efficient manufacturing plants and growing R&D capabilities are now major suppliers of Active Pharmaceutical Ingredients (APIs) and generic drugs across both developed and developing countries. In response to generic competition, innovator companies employ a range of life cycle management techniques, including the launch of 'authorised generics'. The generics segment in Australia will see high growth rates in coming years but the prospect for local manufacturing is bleak. The availability of cheap generics in international markets has put pressure on Pharmaceutical Benefits Scheme (PBS) pricing arrangements, and a new policy direction was announced in November 2006. Lower generics prices will have a negative impact on some incumbent suppliers but industrial renewal policies for the medicines industry in Australia are better focused on higher value R&D activities and niche manufacturing of sophisticated products

NYESO-1/LAGE-1s and PRAME Are Targets for Antigen Specific T Cells in Chondrosarcoma following Treatment with 5-Aza-2-Deoxycitabine

Chondrosarcoma has no proven systemic option in the metastatic setting. The development of a non-cross-resistant strategy, such as cellular immunotherapy using antigen-specific T cells would be highly desirable. NY-ESO-1 and PRAME are members of the Cancer Testis Antigen (CTA) family that have been identified as promising targets for T cell therapy. LAGE-1 is a cancer testis antigen 90% homologous to NY-ESO-1, sharing the 157-165 A*0201 NY-ESO-1 epitope with its transcript variant, LAGE-1s. A number of CTA's have been induced using 5-Aza-2-Deoxycitabine (5-Aza-dC) in other cancers. We sought to evaluate the feasibility of targeting chondrosarcoma tumors using NY-ESO-1/LAGE-1s and PRAME specific T cells using 5-Aza-dC to induce antigen expression.We used 11 flash frozen tumors from the University of Washington tumor bank to test for the expression of NY-ESO-1, PRAME, LAGE-1s and LAGE-1L in chondrosarcoma tumors. Using four chondrosarcoma cell lines we tested the expression of these CTA's with and without 5-Aza-dC treatments. Finally, using NY-ESO-1/LAGE-1s and PRAME specific effectors that we generated from sarcoma patients, we evaluated the ability of these T cells to lyse A*0201 expressing chondrosarcoma cell lines in vitro both with and without 5-Aza-dC treatment.A minority (36%) of chondrosarcoma tumors expressed either NY-ESO-1 or LAGE-1s at >10% of our reference value and none expressed PRAME at that level. However, in all four of the chondrosarcoma cell lines tested, NY-ESO-1 and PRAME expression could be induced following treatment with 5-Aza-dC including in cell lines where expression was absent or barely detectable. Furthermore, NY-ESO-1/LAGE-1s and PRAME specific CD8+ effector T cells were able to specifically recognize and lyse A*0201 expressing chondrosarcoma cell lines following 5-Aza-dC treatment.These data suggest that adoptive immunotherapy in combination with 5-Aza-dC may be a potential strategy to treat unresectable or metastatic chondrosarcoma patients where no proven systemic therapies exist

Clash of pans: pan-Africanism and pan-Anglo-Saxonism and the global colour line, 1919–1945

The article demonstrates both conceptually and empirically that pan-Anglo-Saxonist knowledge networks reconstructed and reimagined an apparently de-racialised, scientific, sober and liberal world order that outwardly abandoned, but did not eradicate the twin phenomena of racism and imperialism. Rather the new liberal (imperial) internationalists, organised in newly formed “think tanks” such as Chatham House and the Council on Foreign Relations, and through their increasingly global elite networks, mounted a top-down battle for minds at home and in the wider world. Operating in state-private elite networks, they drove the movement to manage change and develop a new liberal world order particularly to contain pan-Africanists who combatted the domination and exploitation of Africans worldwide. More broadly, we indicate that the pragmatic response to the extremes of Nazi ideology and a countering movement from the cadres of Asian, African and African American intellectuals, anti-colonial and anti-racist struggles within the national and global context, forced the Anglo-centric elites to promote change, albeit limited

Proinflammatory Phenotype and Increased Caveolin-1 in Alveolar Macrophages with Silenced CFTR mRNA

The inflammatory milieu in the respiratory tract in cystic fibrosis (CF) has been linked to the defective expression of the cystic transmembrane regulator (CFTR) in epithelial cells. Alveolar macrophages (AM), important contibutors to inflammatory responses in the lung, also express CFTR. The present study analyzes the phenotype of human AM with silenced CFTR. Expression of CFTR mRNA and the immature form of the CFTR protein decreased 100-fold and 5.2-fold, respectively, in AM transfected with a CFTR specific siRNA (CFTR-siRNA) compared to controls. Reduction of CFTR expression in AM resulted in increased secretion of IL-8, increased phosphorylation of NF-κB, a positive regulator of IL-8 expression, and decreased expression of IκB-α, the inhibitory protein of NF-κB activation. AM with silenced CFTR expression also showed increased apoptosis. We hypothesized that caveolin-1 (Cav1), a membrane protein that is co-localized with CFTR in lipid rafts and that is related to inflammation and apoptosis in macrophages, may be affected by decreased CFTR expression. Messenger RNA and protein levels of Cav1 were increased in AM with silenced CFTR. Expression and transcriptional activity of sterol regulatory element binding protein (SREBP), a negative transcriptional regulator of Cav1, was decreased in AM with silenced CFTR, but total and free cholesterol mass did not change. These findings indicate that silencing of CFTR in human AM results in an inflammatory phenotype and apoptosis, which is associated to SREBP-mediated regulation of Cav1

Extraction of pure components from overlapped signals in gas chromatography-mass spectrometry (GC-MS)

Gas chromatography-mass spectrometry (GC-MS) is a widely used analytical technique for the identification and quantification of trace chemicals in complex mixtures. When complex samples are analyzed by GC-MS it is common to observe co-elution of two or more components, resulting in an overlap of signal peaks observed in the total ion chromatogram. In such situations manual signal analysis is often the most reliable means for the extraction of pure component signals; however, a systematic manual analysis over a number of samples is both tedious and prone to error. In the past 30 years a number of computational approaches were proposed to assist in the process of the extraction of pure signals from co-eluting GC-MS components. This includes empirical methods, comparison with library spectra, eigenvalue analysis, regression and others. However, to date no approach has been recognized as best, nor accepted as standard. This situation hampers general GC-MS capabilities, and in particular has implications for the development of robust, high-throughput GC-MS analytical protocols required in metabolic profiling and biomarker discovery. Here we first discuss the nature of GC-MS data, and then review some of the approaches proposed for the extraction of pure signals from co-eluting components. We summarize and classify different approaches to this problem, and examine why so many approaches proposed in the past have failed to live up to their full promise. Finally, we give some thoughts on the future developments in this field, and suggest that the progress in general computing capabilities attained in the past two decades has opened new horizons for tackling this important problem