The human topoisomerase I damage response plays a role in apoptosis

Previous studies have shown that human topoisomerase I cleavage complexes form as a response to various DNA damages in vivo, the so called human topoisomerase I “damage response”. It was suggested that this damage response may play a role in DNA repair as well as in apoptosis, but only very few investigations have been done and the significance of the damage response still remains unclear. Here we demonstrate that human topoisomerase I cleavage complexes induced by high doses of UV irradiation are highly stable for up to 48 h. Furthermore, we show that human topoisomerase I cleavage complexes correlate with apoptosis. However, at low UV doses the cleavage complex level was very low and the complexes were repaired. Surprisingly, we found that high levels of stable cleavage complexes were not only found in UV-irradiated cells but also in untreated cells that underwent apoptosis. A possible role of human topoisomerase I in apoptosis is discussed.

Phenytoin metabolism by human cytochrome P450 : involvement of P450 3A and 2C forms in secondary metabolism and drug-protein adduct formation

The anticonvulsant phenytoin (5,5-diphenylhydantoin) provokes a skin rash in 5 to 10% of patients, which heralds the start of an idiosyncratic reaction that may result from covalent modification of normal self proteins by reactive drug metabolites. Phenytoin is metabolized by cytochrome P450 (P450) enzymes primarily to 5-(p-hydroxyphenyl-),5-phenylhydantoin (HPPH), which may be further metabolized to a catechol that spontaneously oxidizes to semiquinone and quinone species that covalently modify proteins. The aim of this study was to determine which P450s catalyze HPPH metabolism to the catechol, proposed to be the final enzymatic step in phenytoin bioactivation. Recombinant human P450s were coexpressed with NADPH-cytochrome P450 reductase in Escherichia coli. Novel bicistronic expression vectors were constructed for P450 2C19 and the three major variants of P450 2C9, i.e., 2C9*1, 2C9*2, and 2C9*3. HPPH metabolism and covalent adduct formation were assessed in parallel. P450 2C19 was the most effective catalyst of HPPH oxidation to the catechol metabolite and was also associated with the highest levels of covalent adduct formation. P450 3A4, 3A5, 3A7, 2C9*1, and 2C9*2 also catalyzed bioactivation of HPPH, but to a lesser extent. Fluorographic analysis showed that the major targets of adduct formation in bacterial membranes were the catalytic P450 forms, as suggested from experiments with human liver microsomes. These results suggest that P450 2C19 and other forms from the 2C and 3A subfamilies may be targets as well as catalysts of drug-protein adduct formation from phenytoin.

Interleukin-1β stimulates human renal fibroblast proliferation and matrix protein production by means of a transforming growth factor-β-dependent mechanism

One of the hallmarks of progressive renal disease is the development of tubulointerstitial fibrosis. This is frequently preceded by macrophage infiltration, raising the possibility that macrophages relay fibrogenic signals to resident tubulointerstitial cells. The aim of this study was to investigate the potentially fibrogenic role of interleukin-1beta (IL-1beta), a macrophage-derived inflammatory cytokine, on cortical fibroblasts (CFs). Primary cultures of human renal CFs were established and incubated for 24 hours in the presence or absence of IL-1beta. We found that IL-1beta significantly stimulated DNA synthesis (356.7% +/- 39% of control, P <.003), fibronectin secretion (261.8 +/- 11% of control, P <.005), collagen type 1 production, (release of procollagen type 1 C-terminal-peptide, 152.4% +/- 26% of control, P <.005), transforming growth factor-beta (TGF-beta) secretion (211% +/- 37% of control, P <.01), and nitric oxide (NO) production (342.8% +/- 69% of control, P <.002). TGF-beta (1 ng/mL) and the phorbol ester phorbol 12-myristate 13-acetate (PMA, 25 nmol/L) produced fibrogenic effects similar to those of IL-1beta. Neither a NO synthase inhibitor (N(G)-methyl-l-arginine, 1 mmol/L) nor a protein kinase C (PKC) inhibitor (bis-indolylmaleimide 1, 1 micromol/L) altered the enhanced level of fibronectin secretion or DNA synthesis seen in response to IL-1beta treatment. However, addition of a TGF-beta-neutralizing antibody significantly reduced IL-1beta-induced fibronectin secretion (IL-1beta + IgG, 262% +/- 72% vs IL-1beta + alphaTGF-beta 156% +/- 14%, P <.02), collagen type 1 production (IL-1beta + IgG, 176% +/- 28% vs IL-1beta + alphaTGF-beta, 120% +/- 14%, P <.005) and abrogated IL-1beta-induced DNA synthesis (245% +/- 49% vs 105% +/- 21%, P <.005). IL-1beta significantly stimulated CF DNA synthesis and production of fibronectin, collagen type 1, TGFbeta, and NO. The fibrogenic and proliferative action of IL-1beta on CF appears not to involve activation of PKC or production of NO but is at least partly TGFbeta-dependent.

Interleukin-1beta induces human proximal tubule cell injury, alpha-smooth muscle actin expression and fibronectin production

BACKGROUND Tubulointerstitial lesions, characterized by tubular injury, interstitial fibrosis and the appearance of myofibroblasts, are the strongest predictors of the degree and progression of chronic renal failure. These lesions are typically preceded by macrophage infiltration of the tubulointerstitium, raising the possibility that these inflammatory cells promote progressive renal disease through fibrogenic actions on resident tubulointerstitial cells. The aim of the present study, therefore, was to investigate the potentially fibrogenic mechanisms of interleukin-1beta (IL-1beta), a macrophage-derived pro-inflammatory cytokine, on human proximal tubule cells (PTC). METHODS Confluent, quiescent, passage 2 PTC were established in primary culture from histologically normal segments of human renal cortex (N = 11) and then incubated in serum- and hormone-free media supplemented with either IL-1beta (0 to 4 ng/mL) or vehicle (control). RESULTS IL-1beta significantly enhanced fibronectin secretion by up to fourfold in a time- and concentration-dependent fashion. This was accompanied by significant (2.5- to 6-fold) increases in alpha-smooth muscle actin (alpha-SMA) expression, transforming growth factor beta (TGF-beta1) secretion, nitric oxide (NO) production, NO synthase 2 (NOS2) mRNA and lactate dehydrogenase (LDH) release. Cell proliferation was dose-dependently suppressed by IL-1beta. NG-methyl-l-arginine (L-NMMA; 1 mmol/L), a specific inhibitor of NOS, blocked NO production but did not alter basal or IL-1beta-stimulated fibronectin secretion. In contrast, a pan-specific TGF-beta neutralizing antibody significantly blocked the effects of IL-1beta on PTC fibronectin secretion (IL-1beta, 268.1 +/- 30.6 vs. IL-1beta+alphaTGF-beta 157.9 +/- 14.4%, of control values, P < 0.001) and DNA synthesis (IL-1beta 81.0 +/- 6.7% vs. IL-1beta+alphaTGF-beta 93.4 +/- 2.1%, of control values, P < 0.01). CONCLUSION IL-1beta acts on human PTC to suppress cell proliferation, enhance fibronectin production and promote alpha-smooth muscle actin expression. These actions appear to be mediated by a TGF-beta1 dependent mechanism and are independent of nitric oxide release.

Mycobacterium abscessus isolated from municipal water : a potential source of human infection

Background Mycobacterium abscessus is a rapidly growing mycobacterium responsible for progressive pulmonary disease, soft tissue and wound infections. The incidence of disease due to M. abscessus has been increasing in Queensland. In a study of Brisbane drinking water, M. abscessus was isolated from ten different locations. The aim of this study was to compare genotypically the M. abscessus isolates obtained from water to those obtained from human clinical specimens. Methods Between 2007 and 2009, eleven isolates confirmed as M. abscessus were recovered from potable water, one strain was isolated from a rainwater tank and another from a swimming pool and two from domestic taps. Seventy-four clinical isolates referred during the same time period were available for comparison using rep-PCR strain typing (Diversilab). Results The drinking water isolates formed two clusters with ≥97% genetic similarity (Water patterns 1 and 2). The tankwater isolate (WP4), one municipal water isolate (WP3) and the pool isolate (WP5) were distinctly different. Patient isolates formed clusters with all of the water isolates except for WP3. Further patient isolates were unrelated to the water isolates. Conclusion The high degree of similarity between strains of M. abscessus from potable water and strains causing infection in humans from the same geographical area, strengthens the possibility that drinking water may be the source of infection in these patients.

Genome-wide DNA methylation analysis of human brain tissue from schizophrenia patients

Recent studies suggest that genetic and environmental factors do not account for all the schizophrenia risk and epigenetics also plays a role in disease susceptibility. DNA methylation is a heritable epigenetic modification that can regulate gene expression. Genome-Wide DNA methylation analysis was performed on post-mortem human brain tissue from 24 patients with schizophrenia and 24 unaffected controls. DNA methylation was assessed at over 485 000 CpG sites using the Illumina Infinium Human Methylation450 Bead Chip. After adjusting for age and post-mortem interval (PMI), 4 641 probes corresponding to 2 929 unique genes were found to be differentially methylated. Of those genes, 1 291 were located in a CpG island and 817 were in a promoter region. These include NOS1, AKT1, DTNBP1, DNMT1, PPP3CC and SOX10 which have previously been associated with schizophrenia. More than 100 of these genes overlap with a previous DNA methylation study of peripheral blood from schizophrenia patients in which 27 000 CpG sites were analysed. Unsupervised clustering analysis of the top 3 000 most variable probes revealed two distinct groups with significantly more people with schizophrenia in cluster one compared to controls (p = 1.74x10-4). The first cluster was composed of 88% of patients with schizophrenia and only 12% controls while the second cluster was composed of 27% of patients with schizophrenia and 73% controls. These results strongly suggest that differential DNA methylation is important in schizophrenia etiology and add support for the use of DNA methylation profiles as a future prognostic indicator of schizophrenia.

Isolation of human lymphatic malformation endothelial cells, their in vitro characterization and in vivo survival in a mouse xenograft model

Human lymphatic vascular malformations (LMs), also known as cystic hygromas or lymphangioma, consist of multiple lymphatic endothelial cell-lined lymph-containing cysts. No animal model of this disease exists. To develop a mouse xenograft model of human LM, CD34NegCD31Pos LM lymphatic endothelial cells (LM-LEC) were isolated from surgical specimens and compared to foreskin CD34NegCD31Pos lymphatic endothelial cells (LECs). Cells were implanted into a mouse tissue engineering model for 1, 2 and 4 weeks. In vitro LM-LECs showed increased proliferation and survival under starvation conditions (P < 0.0005 at 48 h, two-way ANOVA), increased migration (P < 0.001, two-way ANOVA) and formed fewer (P = 0.029, independent samples t test), shorter tubes (P = 0.029, independent samples t test) than foreskin LECs. In vivo LM-LECs implanted into a Matrigel™-containing mouse chamber model assembled to develop vessels with dilated cystic lumens lined with flat endothelium, morphology similar to that of clinical LMs. Human foreskin LECs failed to survive implantation. In LM-LEC implanted chambers the percent volume of podoplaninPos vessels was 1.18 ± 2.24 % at 1 week, 6.34 ± 2.68 % at 2 weeks and increasing to 7.67 ± 3.60 % at 4 weeks. In conclusion, the significantly increased proliferation, migration, resistance to apoptosis and decreased tubulogenesis of LM-LECs observed in vitro is likely to account for their survival and assembly into stable LM-like structures when implanted into a mouse vascularised chamber model. This in vivo xenograft model will provide the basis of future studies of LM biology and testing of potential pharmacological interventions for patients with lymphatic malformations.

Diurnal variations in ocular aberrations of human eyes

Purpose: To investigate the diurnal variations in ocular wavefront aberrations over two consecutive days in young adult subjects. Materials and methods: Measurements of both lower-order (sphero-cylindrical refractive powers) and higher-order (3rd and 4th order aberration terms) ocular aberrations were collected for 30 young adult subjects at ten different times over two consecutive days using a Hartmann-Shack aberrometer. Fifteen subjects were myopic and 15 were emmetropic. Five sets of measurements were collected each day at approximately 3 hourly intervals, with the first measurement taken at ~9 am and the final measurement at ~9 pm. Results: Spherical equivalent refraction (p = 0.029) and spherical aberration (p = 0.043) were both found to undergo significant diurnal variation over the two measurement days. The spherical equivalent was typically found to be at a maximum (i.e. most hyperopic) at the morning measurement, with a small myopic shift of 0.37 ± 0.15 D observed over the course of the day. The mean spherical aberration of all subjects (0.038 ± 0.048 μm) was found to be positive during the day and gradually became more negative into the evening, with a mean amplitude of change of 0.036 ± 0.02 μm. None of the other considered sphero-cylindrical refractive power components or higher-order aberrations exhibited significant diurnal variation over the two days of the experiment (p>0.05). Except for the lower-order astigmatism at 90/180 deg (p = 0.040), there were no significant differences between myopes and emmetropes in the magnitude and timing of the observed diurnal variations (p>0.05). Conclusions: Significant diurnal variations in spherical equivalent and spherical aberration were consistently observed over two consecutive days of measurement. Research and clinical applications requiring precise refractive error and wavefront measurements should take these diurnal changes into account when interpreting wavefront data.

'Too high in human terms' : the costs of high security imprisonment. Review of Imprisoning Resistance by Bree Carlton and Intractable by Bernie Matthews.

The article discusses the issues of resistance; that is resistance by prisoners to the various manifestations of power operating in high security prisons, as well as that of attempted shifts in the regime from physical to psychological control. Other topics highlighted include legitimacy and 'official discourse', mourning and the construction of 'ungrievable lives' and the importance of finding a way out of the cycle of violence, which high security regimes perpetuate.

Species-specific homing mechanisms of human prostate cancer metastasis in tissue engineered bone

The development of effective therapeutic strategies against prostate cancer bone metastases has been impeded by the lack of adequate animal models that are able to recapitulate the biology of the disease in humans. Bioengineered approaches allow researchers to create sophisticated experimentally and physiologically relevant in vivo models to study interactions between cancer cells and their microenvironment under reproducible conditions. The aim of this study was to engineer a morphologically and functionally intact humanized organ bone which can serve as a homing site for human prostate cancer cells. Transplantation of biodegradable tubular composite scaffolds seeded with human mesenchymal progenitor cells and loaded with rhBMP-7 resulted in the development of a chimeric bone construct including a large number of human mesenchymal cells which were shown to be metabolically active and capable of producing extracellular matrix components. Micro-CT analysis demonstrated that the newly formed ossicle recapitulated the morphological features of a physiological organ bone with a trabecular network surrounded by a cortex-like outer structure. This microenvironment was supportive of the lodgement and maintenance of murine haematopoietic cell clusters, thus mimicking a functional organ bone. Bioluminescence imaging demonstrated that luciferase-transduced human PC3 cells reproducibly homed to the humanized tissue engineered bone constructs, proliferated, and developed macro-metastases. This model allows the analysis of interactions between human prostate cancer cells and a functional humanized bone organ within an immuno-incompetent murine host. The system can serve as a reproducible platform to study effects of therapeutics against prostate cancer bone metastases within a humanized microenvironment.

Integrin αvβ3 upregulates integrin-linked kinase expression in human ovarian cancer cells via enhancement of ILK gene transcription

We previously showed that integrin alphavbeta3 overexpression and engagement by its ligand vitronectin increased adhesion, motility, and proliferation of human ovarian cancer cells. In search of differentially regulated genes involved in these tumor biological events, we previously identified the integrin-linked kinase (ILK) to be under control of alphavbeta3. In the present investigation we demonstrated significantly upregulated ILK protein as a function of alphavbeta3 in two ovarian cancer cell lines, OV-MZ-6 and OVCAR-3, and proved co-localization at the surface of alphavbeta3-overexpressing cells adherent to vitronectin. Increase of ILK protein was reflected by enhanced ILK promoter activity, an effect, which we further characterized with regard to transcriptional response elements involved. Abrogation of NF-kappaB/c-rel or p53 binding augmented ILK promoter activity and preserved induction by alphavbeta3. The AP1-mutant exhibited decreased promoter activity but was also still inducible by alphavbeta3. Disruption of the two DNA consensus motifs for Ets proteins led to divergent observations: mutation of the Ets motif at promoter position -462 bp did not significantly alter promoter activity but still allowed response to alphavbeta3. In contrast, disruption of the second Ets motif at position -85 bp did not only lead to slightly diminished promoter activity but also, in that case, abrogated ILK promoter induction by alphavbeta3. Subsequent co-transfection studies with ets-1 in the presence of the second Ets motif led to additional induction of ILK promoter activity. Taken together, these data suggest that ets-1 binding to the second Ets DNA motif strongly contributes to alphavbeta3-mediated ILK upregulation. By increasing ILK as an important integrin-proximal kinase, alphavbeta3 may promote its intracellular signaling and tumor biological processes arising thereof in favor of ovarian cancer metastasis.

Elucidating the host–pathogen interaction between human colorectal cells and invading Enterovirus 71 using transcriptomics profiling

Enterovirus 71 (EV71) is one of the main etiological agents for Hand, Foot and Mouth Disease (HFMD) and has been shown to be associated with severe clinical manifestation. Currently, there is no antiviral therapeutic for the treatment of HFMD patients owing to a lack of understanding of EV71 pathogenesis. This study seeks to elucidate the transcriptomic changes that result from EV71 infection. Human whole genome microarray was employed to monitor changes in genomic profiles between infected and uninfected cells. The results reveal altered expression of human genes involved in critical pathways including the immune response and the stress response. Together, data from this study provide valuable insights into the host–pathogen interaction between human colorectal cells and EV71.

‘I came to this country for a better life’ : factors mediating employment trajectories among young people who migrated to Australia as refugees during adolescence

Located at the intersection of two vulnerable groups in the contemporary labour market, young people who migrate as refugees during adolescence face a unique constellation of opportunities and challenges that shape their employment trajectories. Yet the tendency for research to focus on the early years of refugee settlement means that we have an inadequate understanding the factors that mediate their employment decisions, experiences and outcomes. Based on interviews with 51 young people, this article explores how aspirations, responsibilities, family, education and networks are understood to influence the employment trajectories of adolescent refugee migrants. While this article draws attention to the complex and dynamic range of challenges and constraints that these young people negotiate in the pursuit of satisfying and sustainable employment, what also emerges is an optimistic and determined cohort who, even as they at times unsuccessfully prepare for and navigate the labour market, maintain high hopes for a better life.

The Church of England : Charity Law and Human Rights

This book examines the interface between religion, charity law and human rights. It does so by treating the Church of England and its current circumstances as a timely case study providing an opportunity to examine the tensions that have now become such a characteristic feature of that interface. Firstly, it suggests that the Church is the primary source of canon law principles that have played a formative role in shaping civic morality throughout the common law jurisdictions: the history of their emergence and enforcement by the State in post-Reformation England is recorded and assessed. Secondly, it reveals that of such principles those of greatest weight were associated with matters of sexuality: in particular, for centuries, family law was formulated and applied with regard for the sanctity of the heterosexual marital family which provided the only legally permissible context for any form of sexual relationship. Thirdly, given that history, it identifies and assesses the particular implications that now arise for the Church as a consequence of recent charity law reform outcomes and human rights case law developments: a comparative analysis of religion related case law is provided. Finally, following an outline of the structure and organizational functions of the Church, a detailed analysis is undertaken of its success in engaging with these issues in the context of the Lambeth Conferences, the wider Anglican Communion and in the ill-fated Covenant initiative. From the perspective of the dilemmas currently challenging the moral authority of the Church of England, this book identifies and explores the contemporary ‘moral imperatives’ or red line issues that now threaten the coherence of Christian religions in most leading common law nations. Gay marriage and abortion are among the host of morally charged and deeply divisive topics demanding a reasoned response and leadership from religious bodies. Attention is given to the judicial interpretation and evaluation of these and other issues that now undermine the traditional role of the Church of England. As the interface between religion, charity law and human rights becomes steadily more fractious, with religious fundamentalism and discrimination acquiring a higher profile, there is now a pressing need for a more balanced relationship between those with and those without religious beliefs. This book will be an invaluable aid in starting the process of achieving a triangulated relationship between the principles of canon law, charity law and human rights law.