8 research outputs found
Electrospray mass spectrometry and nuclear magnetic resonance spectroscopy of DNA-interactive agents
No full textThe binding of the antitumour antibiotics pyrindamycin A (duocarmycin C2) and pyrindamycin B (duocarmycin C1) to self-complementary oligonucleotides has been studied using electrospray ionisation mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS). The pyrindamycins bind via non-covalent interactions in the minor groove of DNA with subsequent alkylation of the N3 of adenine (particularly within 5'-AAA and 5'- TTA sequences). The relative stability of adducts with different oligonucleotides can be inferred from the ESI mass spectra. M S / M S spectra of alkylated adducts enable facile identification of the site of alkylation. W e show here that the site(s) of alkylation by the pyrindamycins are strongly influenced by the location of the preferred high affinity binding sites within these short oligonucleotides. Alkylation of guanine by pyrindamycin A is shown to occur only upon binding to single-stranded oligonucleotides, providing further confirmation of the importance of the initial non-covalent interaction in conferring sequence selectivity. These data clearly demonstrate value of using ESI-MS/MS to prescreen ligand-oligonucleotide complexes prior to performing more detailed structural studies. In the second part of this work, the binding of nogalamycin to unusual DNA structures was explored by nuclear magnetic resonance (NMR) spectroscopy. The first of these was an oligonucleotide 5'-d(GCGAAGCACGAAGT)-3', which was designed to form a double hairpin or loop sequence. T w o further oligonucleotides, 5'-d(GTGCGAAGCTAC)-3' and 5'-d(GCTACGAAGTGC)-3' were designed to incorporate a mispaired thymine, which may then form a bulge. The folding of the hairpins was evidenced by anomalous chemical shift values for the sugar H4', H5' and H5" protons (characteristic of the adenine involved in the loop) and the presence of imino protons (protected from exchange by hydrogen bonding interactions in the stem). Nogalamycin formed 1:1 complexes with each of the oligonucleotides with the ligand bound at the TpG step. Upon ligand binding, changes in chemical shift values for the DNA resonances were observed, with the largest differences noted for the nucleotides in the intercalation site. Nogalamycin bound to 5'-d(GCGAAGCACGAAGT)-3' at the nicked site and stabilised the double hairpin structure through co-axial stacking interactions. The intercalation of nogalamycin to 5'-d(GTGCGAAGCTAC)-3' gave rise to an equilibrium in solution between a frame-shifted conformation and bulged conformation. The binding of nogalamycin to 5'-d(GCTACGAAGTGC)-3' stabilised the bulge positioned at T3. Finally, ESI-MS and melting temperature data indicate that the relative stability of the complexes formed with nogalamycin is in the order: 5'-d(GCGAAGCACGAAGT)-3'-Ng > 5'- d(GCTACGAAGTGC)-3'-Ng > 5'-d(GTGCGAAGCTAC)-3'-Ng
Cloning and tissue distribution of novel splice variants of the ovine ghrelin gene
No full textBackground: The ghrelin axis is involved in the regulation of metabolism, energy balance, and the immune, cardiovascular and reproductive systems. The manipulation of this axis has potential for improving economically valuable traits in production animals, and polymorphisms in the ghrelin (GHRL) and ghrelin receptor (GHSR) genes have been associated with growth and carcass traits. Here we investigate the structure and expression of the ghrelin gene (GHRL) in sheep, Ovis aries. Results: We identify two ghrelin mRNA isoforms, which we have designated Δex2 preproghrelin and Δex2,3 preproghrelin. Expression of Δex2,3 preproghrelin is likely to be restricted to ruminants, and would encode truncated ghrelin and a novel C-terminal peptide. Both Δex2 preproghrelin and canonical preproghrelin mRNA isoforms were expressed in a range of tissues. Expression of the Δex2,3 preproghrelin isoform, however, was restricted to white blood cells (WBC; where the wild-type preproghrelin isoform is not co-expressed), and gastrointestinal tissues. Expression of Δex2 preproghrelin and Δex2,3 preproghrelin mRNA was elevated in white blood cells in response to parasitic worm (helminth) infection in genetically susceptible sheep, but not in resistant sheep. Conclusions: The restricted expression of the novel preproghrelin variants and their distinct WBC expression pattern during parasite infection may indicate a novel link between the ghrelin axis and metabolic and immune function in ruminants
Analytical Methods for Allergen Control in Food Processing
No full textFood allergy and food-related anaphylaxis have become a growing public health and food safety issue worldwide [...
The function of the natural antisense ghrelin receptor gene (GHSROS) in cancer
No full textLung cancer is the most common cause of cancer deaths in men and women in the world and the incidence in Australia is rising with our aging population. Survival rates for lung cancer are very poor. We have discovered a new gene that is produced by lung cancer cells and may contribute to the aggressive nature of this disease. We will investigate this gene to determine if it could be a useful target for new therapies for lung cancer and it determine its utility as a biomarker for the severity of the disease.$AUD 493,750.49NHMRC Project GrantsStandard Project Gran
Global proteomic profiling of the membrane compartment of bovine testis cell populations: DOI: 10.5584/jiomics.v3i2.136
No full text
Spermatogonial stem cells hold enormous potential in mammalian reproductive medicine through the preservation of gametes, the restoration of fertility, enhancement of germ-lineage genetic manipulation and the improvement in our understanding of stem cell biology. Here we describe the protein profiles of the membrane compartment of bovine testicular cell isolates which were enriched for germ cells using differential plating. The isolated cells were characterised with antibodies to UCHL1 (previously known as PGP9.5) for type A spermatogonia; DDX4 (previously known as VASA) for germ cells and vimentin for Sertoli cells. Ultracentrifugation techniques were used to specifically isolate cell membranes, with membrane protein identifications significantly increased when compared to whole cell lysates. We utilised the filter-aided sample preparation protocol for improved digestion efficiency of membrane proteins. Using ESI-LC-MS/MS, we compared the proteins present in two cell populations. A total of 1,387 proteins were identified in bovine testis cell isolates, of which 39% were membrane-associated. A total of 64 proteins were differentially expressed in the non-adhered fraction (enriched for undifferentiated germ cells), of which 16 were unique to this cell population and the remaining 48 showed a two-fold change as judged by spectral counting. This analysis revealed a number of candidate germ cell markers including the known markers, DDX4 and UCHL1. The proteomic profiles generated in this study support and complement transcriptomics studies and reinforce the potential of proteomics in identifying and characterising the protein effectors of self-renewal and/or differentiation in stem cells
The Old English Bede: Transmission and Textual History in Anglo-Saxon Manuscripts
No full textAn unknown author translated the Old English version of Bede’s Ecclesiastical History (OEB) around the ninth century. Previous research focused on the text’s authorship, specifically on Mercian linguistic features in its earliest manuscript, rather than the reception and transmission of its manuscripts (Miller, 1890; Whitelock, 1962; Kuhn, 1972). This thesis considers the OEB’s reception and transmission as evident in its copyists’ scribal performances. Conservative and innovative textual variants are identified for the OEB, and scribal behaviour categorised according to the framework devised by Benskin and Laing (1981) in their study of Middle English scribes. A detailed linguistic comparison of OEB witnesses combined with a close examination of the physical manuscripts reveals the working methods of scribes involved in their production. The manuscripts examined are:
Oxford, Bodleian Library Tanner 10 (T)
Oxford, Corpus Christi College 279B (O)
Cambridge, Corpus Christi College 41 (B)
Cambridge, University Library Kk.3.18 (Ca)
Each chapter analyses a particular scribal performance. O’s scribe created a Mischsprache text, combining Mercian and West-Saxon forms, yet conflicting views of what constituted a good text are revealed by O’s producers’ extensive textual corrections. Relict forms in B demonstrate that its exemplar was illegible in places and that the scribe was forced to make several textual repairs. Ca has long been considered a direct copy of O, however my detailed comparison of the two manuscripts reveals that this cannot be the case. Finally, some previously unnoticed and unpublished drypoint annotations to O’s text are presented and explored in the context of other Anglo-Saxon scratched material.
This thesis shows the benefits of examining the OEB from a scribal viewpoint, identifying common modes of scribal behaviour across the medieval period. It proposes a set of features belonging to the original translation, some of which hint at an earlier date of composition than previously supposed
Change in Northumbria : was Aldfrith of Northumbria's reign a period of innovation or did it merely reflect the development of processes already underway in the late seventh century?
No full textThis thesis looks at a period of Northumbrian history when the king was a part Irish, Iona trained scholar. Some have suggested that Aldfrith was assisted to the kingship by the northern victors of the battle of Nechtansmere. It examines processes in the late seventh century to try to identify changes that might have happened during the reign of this king.
The study begins with a wide overview of previous research to establish a basis from which to look for processes and change and also examines the sources available to us, written and archaeological. It then looks at the kingdoms to the north and west and at Aldfrith and the period of his reign. The suggestion is made that Aldfrith acted, with the Church, to cult saints that were Northumbrian and Romanist, as opposed to other options that might have been available. It proposes that the Northumbrians rejected opportunities to develop links with the north and west that may have been open to them. The following chapters then examine processes underway in Northumbria in three general areas; in the use of power, in society, and in the economy.
It concludes that although many processes continued as before, these sped up and in certain areas such as the production of coins, and the consequential development of trade, it was a period of innovation. There is no evidence of a focus to the north and west during Aldfrith’s reign and this has implications for how Aldfrith got to the throne, suggesting that it was with the support of the Northumbrian elite and not through the military strength of the Dál Riata or the Picts. The evidence is that Northumbria increasingly looked south for its influences and is prepared to absorb and implement processes and approaches from southern England, Gaul and Rome
