Development of taxane resistance in a panel of human lung cancer cell lines

Using a selection process designed to reflect clinically relevant conditions, a panel of taxane-selected variants were developed to study further the mechanisms of resistance in lung cancer. Unlike continuous or pulse exposure to high concentrations of chemotherapeutic drugs which yield high resistance and often cross resistance, most variants developed here displayed low level resistance to the selecting drug with slight cross-resistance. Pulsing with taxol resulted in more highly resistant clones (up to 51.4-fold). Analysis of taxol and taxotere in the four major lung cancer cell types showed the taxanes to be more effective against NSCLC (with the exception of SKMES-taxane selected variants) than against the SCLC. Comparison of taxol and taxotere shows that taxol induces higher levels of resistance than taxotere. Further, in taxotere-selected cell lines, the cells are more resistant to taxol than taxotere, suggesting that taxotere may be a superior taxane from a clinical view. Taxol treatment resulted in increased cross-resistance to 5-FU in all classes of lung cancer except DMS-53. The high levels of Pgp in the DMS-53 and selected variant suggests this mechanism is not related to Pgp expression. Analysis of the Pgp and MRP-1 status by combination inhibitory assays and Western blotting showed no consistent relationship between expression of the membrane pumps Pgp or MRP-1 and resistance. However, where high level resistance was seen, the parent cell line expressed Pgp or MRP-1 and was accompanied by increased levels in the variants. Overall we found that the clinically relevant models used here are useful for investigating mechanisms of taxane resistance

RAGE: Exacting a toll on the host in response to polymicrobial sepsis and Listeria monocytogenes

The receptor for advanced glycation endproducts (RAGE) has complex roles in the immune/inflammatory response. RAGE is expressed on monocytes/macrophages, T and B lymphocytes, and dendritic cells. Previous studies illustrated that homozygous RAGE-/- mice subjected to overwhelming bacterial sepsis displayed normal clearance of pathogenic bacteria and significantly increased survival. In this issue of Critical Care, Lutterloh and colleagues confirm these findings and provide evidence that blocking antibodies to RAGE afford similar protection in mice, even when administration of anti-RAGE is delayed by 24 hours. Furthermore, these authors illustrate that deletion of RAGE is remarkably protective in mice infected with the intracellular pathogen Listeria monocytogenes. In this Commentary, we consider these findings and propose possible mechanisms by which RAGE exacts a heavy toll on the host in response to polymicrobial sepsis and L. monocytogenes

Team Teaching project: Facilitating effective co-teaching among co-operating teachers and student teachers on school placement.

Abstract available in text

JAK2V617F promotes replication fork stalling with disease-restricted impairment of the intra-S checkpoint response

Cancers result from the accumulation of genetic lesions, but the cellular consequences of driver mutations remain unclear, especially during the earliest stages of malignancy. The V617F mutation in the JAK2 non-receptor tyrosine kinase (JAK2V617F) is present as an early somatic event in most patients with myeloproliferative neoplasms (MPNs), and the study of these chronic myeloid malignancies provides an experimentally tractable approach to understanding early tumorigenesis. Introduction of exogenous JAK2V617F impairs replication fork progression and is associated with activation of the intra-S checkpoint, with both effects mediated by phosphatidylinositide 3-kinase (PI3K) signaling. Analysis of clonally derived JAK2V617F-positive erythroblasts from MPN patients also demonstrated impaired replication fork progression accompanied by increased levels of replication protein A (RPA)-containing foci. However, the associated intra-S checkpoint response was impaired in erythroblasts from polycythemia vera (PV) patients, but not in those from essential thrombocythemia (ET) patients. Moreover, inhibition of p53 in PV erythroblasts resulted in more gamma-H2Ax (γ-H2Ax)–marked double-stranded breaks compared with in like-treated ET erythroblasts, suggesting the defective intra-S checkpoint function seen in PV increases DNA damage in the context of attenuated p53 signaling. These results demonstrate oncogene-induced impairment of replication fork progression in primary cells from MPN patients, reveal unexpected disease-restricted differences in activation of the intra-S checkpoint, and have potential implications for the clonal evolution of malignancies

Rolling cycle translation of circularized infinite open reading frames; fooling the ribosome

Poster Number 156 ROLLING CYCLE TRANSLATION OF CIRCULARIZED INFINITE OPEN READING FRAMES; FOOLING THE RIBOSOME Alan Costello, National Institute for Cellular Biotechnology, Dublin City University [email protected] Nga Lao, National Institute for Cellular Biotechnology, Dublin City University Niall Barron, National Institute for Bioprocessing Research and Training, University College Dublin Martin Clynes, National Institute for Cellular Biotechnology, Dublin City University Key Words: Circular RNA, Translation, RNA structure, Cell engineering, Chinese Hamster Ovary (CHO) cell Recent. Please click Additional Files below to see the full abstract

Topics in the phonology and morphosyntax of Balinese based on the dialect of Singaraja, North Bali

This study describes features of the phonology, verbal morphology, and morphosyntax of the Balinese of Singaraja, in Buleleng district, north Bali. In chapter two I describe the phoneme repertoire, and their realisations. I give evidence for meaningful postlexical rules, of the kind described by Woodbury (1987). In chapter 3 I show that there is a regular interaction between semantics and phonology at a prelexical level, contra the double articulation hypothesis, and in support of the findings of Fudge (1970). I propose an (ultimately pre-linguistic) explanation for this interaction. The findings support and enrich optimality theory, which seeks to formalise the gradient nature of wellformedness (Prince & Smolensky 1993). The semantic evidence helps explain why non-optimality is tolerated in the phonology. While the phoneme is an important unit of organisation at an intermediate level, it is neither a phonological primitive, nor the largest recurrent phonological unit. I give evidence in chapter 4, and in chapter 7, for complex phonological formants more complex than the phoneme. These can be 'segmental', or involve processes such as reduplication (pace the treatment of reduplication in Prince 1987). In chapter 5 various levels of prosodic organisation are described. The syllable, the foot and the phonological word are each relevant to word-formation processes (though no evidence was found for the mora). I give evidence in this chapter, and in chapters 6 and 8, that all morphemes must be well-formed at foot level during the lexical phonology. These data support, and can be accounted for in terms of, the Prosodic Morphology theories developed by McCarthy and Prince (eg 1990). In chapter 6 constraints on the structure of morphs and morphemes are described. Balinese shows similar cooccurrence restrictions to Arabic (Greenberg 1960, McCarthy 1986) and Javanese (Uhlenbeck 1949, Yip 1989), dispreferring the occurrence of more than one consonant of a given place of articulation, underlyingly. Morphemes showing exceptions to this 'preferred rule' predictably belong to the expressive semantic classes defined in chapter 3. Non-prosodic phonological processes involved in morpheme- and word-formation (including concatenation and morphophonemic alternations) are described in chapter 7. Chapter 8 catalogues the various formal types of reduplication. Two previously undescribed surface reduplication types in Balinese are identified: Foot-reduplication and internal reduplication. As with the restrictions on consonant cooccurrence morpheme-internally (chapter 6), reduplication processes involving vowel overwrite give evidence for a non-linear organisation of phonological structure. A variety of evidence is presented to show that phonological or 'inherent' reduplication is asynchronic process in Balinese, (despite previous claims that there is no evidence for this). Finally, I describe theoretically problematical reduplication processes where nondistinctive material is transferred (cf Steriade 1988), and offer an explanation for them in terms of the Prosodic Morphology theory of McCarthy and Prince. The second part of the thesis describes aspects of the morphosyntax, in particular word formation processes applying to derive verbs in Balinese, and related aspects of clause syntax. After an overview of basic features of morphosyntax and terminology in chapters 9 and 10, I describe in detail the functioning of verbal affixes, in chapters 11 to 13. In chapter 14 I give evidence that Balinese shows 'undergoer primacy' in a variety of ways, at the same time arguing against an ergative analysis of the syntax (pace Blake 1993). My major conclusions relate to the function of verbal morphology, and the way meaning is assigned to lexemes and clauses. The meaning of a given root-affix combination is broadly predictable, once the semantics of the root mopheme is known. To that extent, morphology signals primarily semantic information in Balinese. The Actor/Undergoer distinction which informs the morphological and syntactic patterning of other Austronesian languages (Foley & Van Valin 1984, Durie 1984, 1987) is an important one in Balinese. Prefixes (or their absence) signal whether the subject of both transitive and intransitive verbs is an Actor or an Undergoer (chapter 11). The semantic type of the Undergoer is further specified by verbal suffixing (chapter 12). Although formally Balinese is close to the ideal of an agglutinating language, the meaning of a lexeme is not simply the sum of the meanings of its component morphemes (cfKoch 1990). Meaning assignment is best treated using a model such as those in the word & paradigm tradition (Beard 1988, Aronoff 1994). Affixes are not in themselves meaningful: they serve primarily to signal the presence of meaning elements inherent in the lexemes of which they form part. As such some lexical bases always occur with affixes, even though they have not undergone derivational processes. (I argue against the 'precategorial' analysis of such lexical bases in chapter 10). Where affixes participate in derivational processes, the meaning of the resulting lexeme is assigned by the derivational process as a whole. Indeed, I show that in some cases an adequate account of the meaning conveyed by a given lexeme can only be achieved if discourse and other contextual factors are also taken into account (Chapter 13). The evidence from Balinese supports the view of (for example) Wierzbicka and Givon that transitivity is fundamentally a semantic notion. The primary function of affixing on verbs in Balinese is to signal the presence of entitities in the semantic structure of the lexeme; these may or may not be represented in the syntax, according to contextual and other factors. Semantically and formally transitive verbs thus often behave as though they are syntactically intransitive, and affixes often described as 'transitivising' are so as long as one accepts this semantic interpretation of the term. This approach enables a wider explanatory coverage than previous descriptions of Balinese verb morphology, or of similar facts in related languages (Chung 1976, Kana 1986, Alsagoff 1992)

An examination of the impact of the Internet on modern Western astrology.

Astrology is a feature of modern culture. While the academic study of the culture of astrology is on the increase, virtually no scholarship exists on astrology and the Internet. However a large body of literature exists on the relationship between the Internet and religion, and this literature is used as a framework for the study of astrology and Internet. This research investigates the use of the Internet by modern Western Astrologers, within the context of theories of cyberspace. It looks at how the Internet is being used by astrologers and what effects they believe it can have on astrology and its practice. The research was both quantitative and qualitative. Questionnaires were issued at astrological conferences in the United Kingdom and the United States of America. In addition sixty-five astrologers were interviewed. In the 1990s a body of literature was produced that associated the physical Internet with the virtual world of cyberspace. From this literature came claims of cyberspace as dualistic or Cartesian. My research was informed by theories of dualism inherited from the classical world, and by previous arguments that astrology is dualistic. The thesis concludes that the majority of astrologers have a dualistic view of the Internet and cyberspace; the online world of cyberspace is viewed as a mental arena in contrast to the offline, physical world. A highly positive use of the Internet is the growth of online astrological communities; connections can be made with astrologers in different parts of the world. The Internet is perceived as a source of vast quantities of astrological information of varying quality. In the views of the astrologers poor quality astrological information can have a detrimental effect on the practice of astrology in the modern Western world

Differential protein expression following low temperature culture of suspension CHO-K1 cells

<p>Abstract</p> <p>Background</p> <p>To ensure maximal productivity of recombinant proteins (rP) during production culture it is typical to encourage an initial phase of rapid cell proliferation to achieve high biomass followed by a stationary phase where cellular energies are directed towards production of rP. During many such biphasic cultures, the initial phase of rapid cell growth at 37°C is followed by a growth arrest phase induced through reduction of the culture temperature. Low temperature induced growth arrest is associated with many positive phenotypes including increased productivity, sustained viability and an extended production phase, although the mechanisms regulating these phenotypes during mild hypothermia are poorly understood.</p> <p>Results</p> <p>In this study differential protein expression in suspension CHO-K1 cells was investigated following a reduction of the culture temperature from 37°C to 31°C in comparison to standard batch culture maintained at 37°C using 2D-DIGE (Fluorescence 2-D Difference Gel Electrophoresis) and mass spectrometry (MS). There is only limited proteomic analysis of suspension-grown CHO cells describing a direct comparison of temperature shifted versus non-temperature shifted cultures using 2D-DIGE. This investigation has enabled the identification of temperature-dependent as well as temperature-independent proteomic changes. 201 proteins were observed as differentially expressed following temperature shift, of which 118 were up regulated. Of the 53 proteins identified by MALDI-ToF MS, 23 were specifically differentially expressed upon reduction of the culture temperature and were found related to a variety of cellular functions such as regulation of growth (HNRPC), cap-independent translation (EIF4A), apoptosis (importin-α), the cytoskeleton (vimentin) and glycoprotein quality control (alpha glucosidase 2).</p> <p>Conclusion</p> <p>These results indicate the extent of the temperature response in CHO-K1 cells and suggest a number of key regulatory proteins and pathways that are involved in modulating the response of cells to mild hypothermia. Regulation of these identified proteins and pathways could be useful for future approaches to engineer CHO cells for improved recombinant protein production.</p

Bromodeoxyuridine induces keratin protein synthesis at a posttranscriptional level in human lung tumour cell lines

Keratin intermediate filaments are formed in epithelial cells in a cell- and tissue-specific manner, but much remains unknown regarding the mechanisms which control the synthesis of these proteins. We examined the effect of the differentiation modulation agent, bromodeoxyuridine (BrdU), on two human keratin-negative (by immunocytochemistry) lung cell lines, DLKP and H82, and showed immunohistochemically that treatment with 10 µM BrdU over 7 days induced K8 and K18 protein synthesis in both lines. Immunoprecipitation and Western blot analyses revealed low levels of K8 and K18 proteins in untreated cell homogenates. These levels increased following treatment with BrdU for 7 days. K8 and K18 mRNAs were detected by Northern blot and reverse transcriptase polymerase chain reaction analyses in both lines before BrdU treatment, but no increase in mRNA levels was observed in either cell line over 21 days of treatment. This suggests, firstly, that keratin synthesis is normally blocked at a posttranscriptional level in DLKP and H82 cells, and secondly, that BrdU can reverse this block. A549 is a human lung cell line which contains K8 and K18 proteins. Treatment with BrdU increased K8 and K18 protein levels in these cells. No corresponding increase in K8 mRNA levels occurred, while an apparent increase in K18 mRNA levels was detected. HL-60 is a leukaemic cell line of haematopoietic rather than epithelial lineage which contains K8 and K18 mRNA transcripts prior to BrdU treatment, but does not contain keratin proteins. Again, K8 and K18 mRNA levels remained unchanged during BrdU treatment. However, neither K8 nor K18 proteins were detected following treatment, although BrdU is known to alter expression of other genes in HL-60 cells. BrdU thus appears to act at a posttranscriptional level and in an epithelial-specific manner to reverse a block in keratin synthesis in keratinnegative lung cancer cells and increase synthesis in keratin-positive lung cancer cells. This may represent a regulatory step in early lung development or a mechanism whereby tumour cells downregulate expression of a differentiated phenotype