Direct evidence for base-mediated decomposition of alkyl hydroperoxides (ROOH) in the gas phase

Alkyl hydroperoxides (ROOH) are attributed a key role in the biochemical oxidation of lipids during oxidative stress.1 In this chemistry ROOH compounds, where the R groups are unsaturated fatty acids, are viewed as transient ntermediates which are readily degraded, due to the lability of the RO-OH bond, to yield potentially genotoxic aldehydes and ketones.2 Generally, the decomposition of alkyl hydroperoxides is thought to be mediated by radical abstraction or electron transfer processes usually involving enzymes, transition metals, or recently, Vitamin C.3 In this paper we present the first unambiguous experimental and computational evidence for base-mediated heterolytic decomposition of simple alkyl hydroperoxides by the mechanism outlined in Scheme 1.

Staurosporine augments EGF-mediated EMT in PMC42-LA cells through actin depolymerisation, focal contact size reduction and Snail1 induction : a model for cross-modulation

Background A feature of epithelial to mesenchymal transition (EMT) relevant to tumour dissemination is the reorganization of actin cytoskeleton/focal contacts, influencing cellular ECM adherence and motility. This is coupled with the transcriptional repression of E-cadherin, often mediated by Snail1, Snail2 and Zeb1/δEF1. These genes, overexpressed in breast carcinomas, are known targets of growth factor-initiated pathways, however it is less clear how alterations in ECM attachment cross-modulate to regulate these pathways. EGF induces EMT in the breast cancer cell line PMC42-LA and the kinase inhibitor staurosporine (ST) induces EMT in embryonic neural epithelial cells, with F-actin de-bundling and disruption of cell-cell adhesion, via inhibition of aPKC. Methods PMC42-LA cells were treated for 72 h with 10 ng/ml EGF, 40 nM ST, or both, and assessed for expression of E-cadherin repressor genes (Snail1, Snail2, Zeb1/δEF1) and EMT-related genes by QRT-PCR, multiplex tandem PCR (MT-PCR) and immunofluorescence +/- cycloheximide. Actin and focal contacts (paxillin) were visualized by confocal microscopy. A public database of human breast cancers was assessed for expression of Snail1 and Snail2 in relation to outcome. Results When PMC42-LA were treated with EGF, Snail2 was the principal E-cadherin repressor induced. With ST or ST+EGF this shifted to Snail1, with more extreme EMT and Zeb1/δEF1 induction seen with ST+EGF. ST reduced stress fibres and focal contact size rapidly and independently of gene transcription. Gene expression analysis by MT-PCR indicated that ST repressed many genes which were induced by EGF (EGFR, CAV1, CTGF, CYR61, CD44, S100A4) and induced genes which alter the actin cytoskeleton (NLF1, NLF2, EPHB4). Examination of the public database of breast cancers revealed tumours exhibiting higher Snail1 expression have an increased risk of disease-recurrence. This was not seen for Snail2, and Zeb1/δEF1 showed a reverse correlation with lower expression values being predictive of increased risk. Conclusion ST in combination with EGF directed a greater EMT via actin depolymerisation and focal contact size reduction, resulting in a loosening of cell-ECM attachment along with Snail1-Zeb1/δEF1 induction. This appeared fundamentally different to the EGF-induced EMT, highlighting the multiple pathways which can regulate EMT. Our findings add support for a functional role for Snail1 in invasive breast cancer.

Bimolecular interaction of Insulin-Like Growth Factor (IGF) binding protein-2 with αvβ3 negatively modulates IGF-I-Mediated migration and tumor growth

Both the integrin and insulin-like growth factor binding protein (IGFBP) families independently play important roles in modulating tumor cell growth and progression. We present evidence for a specific cell surface localization and a bimolecular interaction between the αvβ3 integrin and IGFBP-2. The interaction, which could be specifically perturbed using vitronectin and αvβ3 blocking antibodies, was shown to modulate IGF-mediated cellular migration responses. Moreover, this interaction was observed in vivo and correlated with reduced tumor size of the human breast cancer cells, MCF-7β3, which overexpressed the αvβ3 integrin. Collectively, these results indicate that αvβ3 and IGFBP-2 act cooperatively in a negative regulatory manner to reduce tumor growth and the migratory potential of breast cancer cells.

Antisense-mediated suppression of hyaluronan synthase 2 inhibits the tumorigenesis and progression of breast cancer

The progression of several cancers is correlated with the increased synthesis of the glycosaminoglycan, hyaluronan. Hyaluronan is synthesized at the plasma membrane by various isoforms of hyaluronan synthases (HAS). The importance of HAS2 expression in highly invasive breast cancer was characterized by the antisense inhibition of HAS2 (ASHAS2). The effect of HAS2 inhibition on cell proliferation, migration, hyaluronan metabolism, and receptor status was characterized in vitro, whereas the effect on tumorigenicity and metastasis was established in vivo. HAS2 inhibition resulted in a 24-hour lag in proliferation that was concomitant to transient arrest of 79% of the cell population in G 0-G1. Inhibition of HAS2 did not alter the expression of the other HAS isoforms, whereas hyaluronidase (HYAL2) and the hyaluronan receptor, CD44, were significantly down-regulated. ASHAS2 cells accumulated greater amounts of high molecular weight hyaluronan (>10,000 kDa) in the culture medium, whereas mock and parental cells liberated less hyaluronan of three distinct molecular weights (100, 400, and 3,000 kDa). The inhibition of HAS2 in the highly invasive MDA-MB-231 breast cancer cell line inhibited the initiation and progression of primary and secondary tumor formation following s.c. and intracardiac inoculation into nude mice, whereas controls readily established both primary and secondary tumors. The lack of primary and secondary tumor formation was manifested by increased survival times where ASHAS2 animals survived 172% longer than the control animals. Collectively, these unique results strongly implicate the central role of HAS2 in the initiation and progression of breast cancer, potentially highlighting the codependency between HAS2, CD44, and HYAL2 expression. ©2005 American Association for Cancer Research.

Remodeling of purinergic receptor-mediated Ca 2+ signaling as a consequence of EGF-induced epithelial-mesenchymal transition in breast cancer cells

Background The microenvironment plays a pivotal role in tumor cell proliferation, survival and migration. Invasive cancer cells face a new set of environmental challenges as they breach the basement membrane and colonize distant organs during the process of metastasis. Phenotypic switching, such as that which occurs during epithelial-mesenchymal transition (EMT), may be associated with a remodeling of cell surface receptors and thus altered responses to signals from the tumor microenvironment. Methodology/Principal Findings We assessed changes in intracellular Ca 2+ in cells loaded with Fluo-4 AM using a fluorometric imaging plate reader (FLIPR TETRA) and observed significant changes in the potency of ATP (EC 50 0.175 μM (-EGF) versus 1.731 μM (+EGF), P<0.05), and the nature of the ATP-induced Ca 2+ transient, corresponding with a 10-fold increase in the mesenchymal marker vimentin (P<0.05). We observed no change in the sensitivity to PAR2-mediated Ca 2+ signaling, indicating that these alterations are not simply a consequence of changes in global Ca 2+ homeostasis. To determine whether changes in ATP-mediated Ca 2+ signaling are preceded by alterations in the transcriptional profile of purinergic receptors, we analyzed the expression of a panel of P2X ionotropic and P2Y metabotropic purinergic receptors using real-time RT-PCR and found significant and specific alterations in the suite of ATP-activated purinergic receptors during EGF-induced EMT in breast cancer cells. Our studies are the first to show that P2X 5 ionotropic receptors are enriched in the mesenchymal phenotype and that silencing of P2X 5 leads to a significant reduction (25%, P<0.05) in EGF-induced vimentin protein expression. Conclusions The acquisition of a new suite of cell surface purinergic receptors is a feature of EGF-mediated EMT in MDA-MB-468 breast cancer cells. Such changes may impart advantageous phenotypic traits and represent a novel mechanism for the targeting of cancer metastasis.

Transformation in educational practices through STEM

The need for strong science, technology and innovation linkages between Higher Education Institutions (HEIs) and industries is a pivotal point for middle-income countries in their endeavor to enhance human capital in socioeconomic development. Currently, the University-Industry partnerships are at an infant stage in Sri Lankan higher education context. Technological maturity and effective communication skills are contributing factors for an efficient graduate profile. Also, expanding internship programs in particular for STEM disciplines provide work experience to students that would strengthen the relevance of higher education programs. This study reports historical overviews and current trends in STEM education in Sri Lanka. Emphasis will be drawn to recent technological and higher education curricular reforms. Data from the last 10 years were extracted from the higher education sector and Ministry of Higher Education Policy portfolios. Associations and trend analysis of the sector growth were compared with STEM existence, merger and predicted augmentations. Results were depicted and summarised based on STEM streams and disciplines. It was observed that the trend of STEM augmentation in the Sri Lankan Higher Education context is growing at a slow but steady pace. Further analysis with other sectors in particular, Industry information, would be useful and a worthwhile exercise.

Reduced excision repair cross-complementing 1 expression associates with enhanced papilloma formation and fibroblast transformation after genetic disruption of secreted protein acidic and rich in cysteine

SPARC (secreted protein acidic and rich in cysteine)/ osteonectin/BM-40 is a matricellular protein implicated in development, cell transformation and tumorigenesis. We have examined the role of SPARC in cell transformation induced chemically with 7,12-dimethylbenz[a]anthracene (DMBA) and 12- tetradecanoylphorbol-13-acetate (TPA) in embryonic fibroblasts and in the skin of mice. Embryonic fibroblasts from SPARCnull mice showed increases in cell proliferation, enhanced sensitivity to DMBA and a higher number of DMBA/TPA-induced transformation foci. The number of DMBA-DNA adducts was 9 times higher in SPARCnull fibroblasts and their stability was lower than wild-type fibroblasts, consistent with a reduction in excision repair cross-complementing 1 the nucleotide excision repair enzyme in these cells. The SPARCnull mice showed an increase in both the speed and number of papillomas forming after topical administration of DMBA/TPA to the skin. These papillomas showed reduced growth and reduced progression to a more malignant phenotype, indicating that the effect of SPARC on tumorigenesis depends upon the transformation stage and/or tissue context. These data reinforce a growing number of observations in which SPARC has shown opposite effects on different tumor types/stages.

Type I collagen abrogates the clathrin-mediated internalization of membrane type 1 matrix metalloproteinase (MT1-MMP) via the MT1-MMP hemopexin domain

Type I collagen (Col I)-stimulated matrix metalloproteinase-2 (MMP-2) activation via membrane type 1 MMP (MT1-MMP) involves both a transcriptional increase in MT1-MMP expression and a nontranscriptional response mediated by preexisting MT1-MMP. In order to identify which MT1-MMP domains were required for the nontranscriptional response, MCF-7 cells that lack endogenous MT1-MMP were transfected with either wild type or domain mutant MT1-MMP constructs. We observed that mutant constructs lacking the MT1-MMP cytoplasmic tail were able to activate MMP-2 in response to Col I but not a construct lacking the MT1-MMP hemopexin domain. Col I did not alter total MT1-MMP protein levels; nor did it appear to directly induce MT1-MMP oligomerization. Col I did, however, redistribute preexisting MT1-MMP to the cell periphery compared with unstimulated cells that displayed amore diffuse staining pattern. In addition, Col I blocked the internalization of MT1-MMP in a dynamin-dependent manner via clathrin-coated pit-mediated endocytosis. This mechanism of impaired internalization is different from that reported for concanavalin A, since it is not mediated by the cytoplasmic tail of MT1-MMP but rather by the hemopexin domain. In summary, upon Col I binding to its cell surface receptor, MT1-MMP internalization via clathrin-coated pit-mediated endocytosis is impaired through interactions with the hemopexin domain, thereby regulating its function and ability to activate MMP-2.

Transmembrane/cytoplasmic domain-mediated membrane type 1-matrix metalloprotease docking to invadopodia is required for cellinvasion

The invasion of human malignant melanoma cells into the extracellular matrix (ECM) involves the accumulation of proteases at sites of ECM degradation where activation of matrix metalloproteases (MMP) occurs. Here, we show that when membrane type 1 MMP (MT-MMP) was overexpressed in RPMI7951 human melanoma cells, the cells made contact with the ECM, activated soluble and ECM-bound MMP-2, and degraded and invaded the ECM. Further experiments demonstrated the importance of localization of the MT-MMP to invadopodia. Overexpression of MT-MMP without invadopodial localization caused activation of soluble MMP-2, but did not facilitate ECM degradation or cell invasiveness. Up-regulation of endogenous MT-MMP with concanavalin A caused activation of MMP-2. However, concanavalin A treatment prevented invadopodial localization of MT-MMP and ECM degradation. Neither a truncated MT-MMP mutant lacking transmembrane (TM) and cytoplasmic domains (ΔTM(MT-MMP)), nor a chimeric MT-MMP containing the interleukin 2 receptor α chain (IL-2R) TM and cytoplasmic domains (ΔTM(MT-MMP)/TM(IL-2R)) were localized to invadopodia or exhibited ECM degradation. Furthermore, a chimera of the TM/cytoplasmic domain of MT-MMP (TM(MT-MMP)) with tissue inhibitor of MMP 1 (TIMP-1/TM(MT- MMP)) directed the TIMP-1 molecule to invadopodia. Thus, the MT-MMP TM/cytoplasmic domain mediates the spatial organization of MT-MMP into invadopodia and subsequent degradation of the ECM.

ST7-mediated suppression of tumorigenicity of prostate cancer cells is characterized by remodeling of the extracellular matrix

Multiple lines of evidence have provided compelling evidence for the existence of a tumor suppressor gene (TSG) on chromosome 7q31.1. ST7 may be the target of this genetic instability but its designation as a TSG is controversial. In this study, we show that, functionally, ST7 behaves as a tumor suppressor in human cancer. ST7 suppressed growth of PC-3 prostate cancer cells inoculated subcutaneously into severe combined immunodeficient mice, and increased the latency of tumor detection from 13 days in control tumors to 23 days. Re-expression of ST7 was also associated with suppression of colony formation under anchorage-independent conditions in MDA-MB-231 breast cancer cells and ST7 mRNA expression was downregulated in 44% of primary breast cancers. Expression profiling of PC-3 cells revealed that ST7 predominantly induces changes in genes involved in re-modeling the extracellular matrix such as SPARC, IGFBP5 and several matrix metalloproteinases. These data indicate that ST7 may mediate tumor suppression through modification of the tumor microenvironment.

The type I collagen induction of MT1-MMP-mediated MMP-2 activation is repressed by αVβ3 integrin in human breast cancer cells

The influence of αVβ3 integrin on MT1-MMP functionality was studied in human breast cancer cells of differing β3 integrin status. Overexpression of β3 integrin caused increased cell surface expression of αV integrin and increased cellular adhesion to extracellular matrix (ECM) substrates in BT-549, MDA-MB-231 and MCF-7 cells. β3 integrin expression also enhanced the migration of breast cancer cells on ECM substrates and enhanced collagen gel contraction. In vivo, αVβ3 cooperated with MT1-MMP to increase the growth of MCF-7 cells after orthotopic inoculation in immunocompromised mice, but had no influence on in vitro proliferation. Despite these stimulatory effects, overexpression of β3 integrin suppressed the type I collagen (Col I) induced MMP-2 activation in all breast cancer cell lines analyzed. This was also evident in extracts from the MCF-7 tumors in vivo, where MMP-2 activation was stimulated by MT1-MMP transfection, but attenuated with β3 integrin expression. Although our studies confirm important biological effects of αVβ3 integrin on enhancing cell adhesion and migration, ECM remodeling and tumor growth, β3 integrin caused reduced MMP-2 activation in response to Col I in vitro, which appears to be physiologically relevant, as it was also seen in tumor xenografts in vivo. The reduction of MMP-2 activation (and thus MT1-MMP activity) by αVβ3 in response to Col I may be important in scenarios where cells which are activated for matrix degradation need to preserve some pericellular collagen, perhaps as a substrate for cell adhesion and migration, thus maintaining a balanced level of proteolysis required for efficient tumor growth.

Self assembly of plasmonic core-satellite nano-assemblies mediated by hyperbranched polymer linkers

The morphology of plasmonic nano-assemblies has a direct influence on optical properties, such as localised surface plasmon resonance (LSPR) and surface enhanced Raman scattering (SERS) intensity. Assemblies with core-satellite morphologies are of particular interest, because this morphology has a high density of hot-spots, while constraining the overall size. Herein, a simple method is reported for the self-assembly of gold NPs nano-assemblies with a core-satellite morphology, which was mediated by hyperbranched polymer (HBP) linkers. The HBP linkers have repeat units that do not interact strongly with gold NPs, but have multiple end-groups that specifically interact with the gold NPs and act as anchoring points resulting in nano-assemblies with a large (~48 nm) core surrounded by smaller (~15 nm) satellites. It was possible to control the number of satellites in an assembly which allowed optical parameters such as SPR maxima and the SERS intensity to be tuned. These results were found to be consistent with finite-difference time domain (FDTD) simulations. Furthermore, the multiplexing of the nano-assemblies with a series of Raman tag molecules was demonstrated, without an observable signal arising from the HBP linker after tagging. Such plasmonic nano-assemblies could potentially serve as efficient SERS based diagnostics or biomedical imaging agents in nanomedicine.

DDT resistance and transformation by different microbial strains isolated from DDT-contaminated soils and compost materials

Bioremediation is a potential option to treat 1, 1, 1-trichloro-2, 2 bis (4-chlorophenyl) ethane (DDT) contaminated sites. In areas where suitable microbes are not present, the use of DDT resistant microbial inoculants may be necessary. It is vital that such inoculants do not produce recalcitrant breakdown products e.g. 1, 1-dichloro-2, 2-bis (4-chlorophenyl) ethylene (DDE). Therefore, this work aimed to screen DDT-contaminated soil and compost materials for the presence of DDT-resistant microbes for use as potential inoculants. Four compost amended soils, contaminated with different concentrations of DDT, were used to isolate DDT-resistant microbes in media containing 150 mg I -1 DDT at three temperatures (25, 37 and 55°C). In all soils, bacteria were more sensitive to DDT than actinomycetes and fungi. Bacteria isolated at 55°C from any source were the most DDT sensitive. However DDT-resistant bacterial strains showed more promise in degrading DDT than isolated fungal strains, as 1, 1-dichloro 2, 2-bis (4-chlorophenyl) ethane (DDD) was a major bacterial transformation product, while fungi tended to produce more DDE. Further studies on selected bacterial isolates found that the most promising bacterial strain (Bacillus sp. BHD-4) could remove 51% of DDT from liquid culture after 7 days growth. Of the amount transformed, 6% was found as DDD and 3% as DDE suggesting that further transformation of DDT and its metabolites occurred.

The transformation of bureaucracy? Structural change in the Commonwealth public service 1983-93

Commentators have predicted bureaucratic organisations would undergo substantial change as a result of social and economic pressures. We ask whether reforms to the Australian public service over the 1983–93 period exemplify this process. We use the methods of organisational analysis to characterise the direction of change, basing our assessment on the standard structural variables of complexity, formalisation and centralisation, together with a cultural variable. We find evidence that, overall, departments of state in the APS were becoming less bureaucratic in their structure, culture and internal function in the 1983–93 period. However, the effect was not uniform across departments, or unambiguous — formalisation, for example, increased in some respects and decreased in others. Centralisation increased overall, despite devolution of some decision-making.