The expression of ADAM-9 and -10 in prostate cancer and their regulation by dihydrotestosterone, insulin-like growth Factor-1 and epidermal growth factor

Prostrate Cancer(PCa)is the most common cause of cancer death amongst Western males. PCa occurs in two distinct stages. In its early stage, growth and development is dependent primarily on male sex hormones (androgens) such as testosterone, although other growth factors have roles maintaining PCa cell survival in this stage. In the later stage of PCa development, growth and.maintenance is independent of androgen stimulation and growth factors including Insulin-like Growth Factor -1 (IGf.:·l) and Epidermal Growth Factor (EGF) are thought to have more crucial roles in cell survival and PCa progression. PCa, in its late stages, is highly aggressive and metastatic, that is, tumorigenic cells migrate from the primary site of the body (prostate) and travel via the systemic and lymphatic circulation, residing and colonising in the bone, lymph node, lung, and in more rare cases, the brain. Metastasis involves both cell migration and tissue degradation activities. The degradation of the extracellular matrix (ECM), the tissue surrounding the organ, is mediated in part by members of a family of 26 proteins called the Matrix Metalloproteases (MMPs), whilst ceil adhesion molecules, of which proteins known as Integrins are included, mediate ce11 migration. A family of proteins known as the ADAMs (A Disintegrin . And Metalloprotease domain) were a recently characterised family at the commencement of this study and now comprise 34 members. Because of their dual nature, possessing an active metaiioprotease domain, homologous to that of the MMPs, and an integrin-binding domain capable of regulating cell-cell and cell-ECM contacts, it was thought likely that members of the ADAMs family may have implications for the progression of aggressive cancers such as those ofthe prostate. This study focussed on two particular ADAMs -9 and -10. ADAM-9 has an active metalloprotease domain, which has been shown to degrade constituents of the ECM, including fibronectin, in vitro. It also has an integrin-binding capacity through association with key integrins involved in PCa progression, such as a6~1. ADAM-10 has no such integrin binding activities, but its bovine orthologue, MADM, is able to degrade coHagen type IV, a major component of basement membranes. It is likely human ADAM-10 has the same activity. It is also known to cleave Ll -a protein involved in cell anchorage activities - and collagen type XVII - which is a principal component of the hemidesmosomes of cellular tight junctions. The cleavage of these proteins enables the cell to be released from the surrounding environment and commence migratory activities, as required in metastasis. Previous studies in this laboratory showed the mRNA expression of the five ADAMs -9,- 10, -11, -15 and -17 in PCa cell lines, characteristic of androgen-dependent and androgen independent disease. These studies were furthered by the characterisation of AD AM-9, -10 and -17 mRNA regulation by Dihydrotestosterone (DHT) in the androgen-responsive cell line (LNCaP). ADAM-9 and -10 mRNA levels were elevated in response to DHT stimulation. Further to these observations, the expression of ADAM-9 and -10 was shown in primary prostate biopsies from patients with PCa. ADAM-1 0 was expressed in the cytoplasm and on the ceH membrane in epithelial and basal cells ofbenign prostate glands, but in high-grade PCa glands, ADAM-I 0 expression was localised to the nucleus and its expression levels appeared to be elevated when compared to low-grade PCa glands. These studies provided a strong background for the hypothesis that ADAM-9 and -10 have key roles in the development ofPCa and provided a basis for further studies.The aims of this study were to: 1) characterise the expression, localisation and levels, of ADAM-9 and -10 mRNA and protein in cell models representing characteristics of normal through androgen-dependent to androgen-independent PCa, as well as to expand the primary PCa biopsy data for ADAM-9 and ADAM-10 to encompass PCa bone metastases 2) establish an in vitro cell system, which could express elevated levels of ADAM-1 0 so that functional cell-based assays such as cell migration, invasion and attachment could be carried out, and 3) to extend the previous hormonal regulation data, to fully characterise the response of ADAM-9 and -10 mRNA and protein levels to DHT, IGF-1, DHT plus IGF-1 and EGF in the hormonal/growth factor responsive cell line LNCaP. For aim 1 (expression of ADAM-9 and -10 mRNA and protein), ADAM-9 and -10 mRNA were characterised by R T -PCR, while their protein products were analysed by Western blot. Both ADAM-9 and -10 mRNA and protein were expressed at readily detectable levels across progressively metastatic PCa cell lines model that represent characteristics of low-grade,. androgen-dependent (LNCaP and C4) to high-grade, androgen-independent (C4-2 and C4-2B) PCa. When the non-tumorigenic prostate cell line RWPE-1 was compared with the metastatic PCa cell line PC-3, differential expression patterns were seen by Western blot analysis. For ADAM-9, the active form was expressed at higher levels in RWPE-1, whilst subcellular fractionation showed that the active form of ADAM-9 was predominantly located in the cell nucleus. For ADAM-I 0, in both of the cell Jines, a nuclear specific isoform of the mature, catalytically active ADAM-I 0 was found. This isoforrn differed by -2 kDa in Mr (smaller) than the cytoplasmic specific isoform. Unprocessed ADAM-I 0 was readily detected in R WPE-1 cell lines but only occasionally detected in PC-3 cell lines. Immunocytochemistry using ADAM-9 and -10 specific antibodies confirmed nuclear, cytoplasmic and membrane expression of both ADAMs in these two cell lines. To examine the possibility of ADAM-9 and -10 being shed into the extracellular environment, membrane vesicles that are constitutively shed from the cell surface and contain membrane-associated proteins were collected from the media of the prostate cell lines RWPE-1, LNCaP and PC-3. ADAM-9 was readily detectable in RWPE- 1 and LNCaP cell membrane vesicles by Western blot analysis, but not in PC-3 cells, whilst the expression of ADAM-I 0 was detected in shed vesicles from each of these prostate cell lines. By Laser Capture Microdissection (LCM), secretory epithelial cells of primary prostate gland biopsies were isolated from benign and malignant glands. These secretory cells, by Western blot analysis, expressed similar Mr bands for ADAM-9 and -10 that were found in PCa cell lines in vitro, indicating that the nuclear specific isoforrn of ADAM-I 0 was present in PCa primary tumours and may represent the predominantly nuclear form of ADAM-I 0 expression, previously shown in high-grade PCa by immunohistochemistry (IHC). ADAM-9 and -10 were also examined by IHC in bone metastases taken from PCa patients at biopsy. Both ADAMs could be detected at levels similar to those shown for Prostate Specific Antigen (PSA) in these biopsies. Furthermore, both ADAM-9 and -10 were predominantly membrane- bound with occasional nuclear expression. For aim 2, to establish a cell system that over-expressed levels of ADAM-10, two fulllength ADAM-I 0 mammalian expression vectors were constructed; ADAM-I 0 was cloned into pcDNA3.1, which contains a CMV promoter, and into pMEP4, containing an inducible metallothionine promoter, whose activity is stimulated by the addition of CdC}z. The efficiency of these two constructs was tested by way of transient transfection in the PCa cell line PC-3, whilst the pcDNA3.1 construct was also tested in the RWPE-1 prostate cell line. Resultant Western blot analysis for all transient transfection assays showed that levels of ADAM-I 0 were not significantly elevated in any case, when compared to levels of the housekeeping gene ~-Tubulin, despite testing various levels of vector DNA, and, for pMEP4, the induction of the transfected cell system with different degrees of stimulation with CdCh to activate the metallothionine promoter post-transfection. Another study in this laboratory found similar results when the same full length ADAM-10 sequence was cloned into a Green Fluorescent Protein (GFP) expressing vector, as no fluorescence was observed by means of transient tran sfection in the same, and other, PCa cell lines. It was hypothesised that the Kozak sequence included in the full-length construct (human ADAMI 0 naturally occurring sequence) is not strong enough to initiate translation in an artificial system, in cells, which, as described in Aim 1, are already expressing readily detectable levels of endogenous ADAM-10. As a result, time constraints prevented any further progress with Aim 2 and functional studies including cell attachment, invasion and migration were unable to be explored. For Aim 3, to characterise the response of ADAM-9 and -10 mRNA and protein levels to DHT, IGF-1, DHT plus IGF-1 and EGF in LNCaP cells, the levels of ADAM-9 and -10 mRNA were not stimulated by DHT or IGF-I alone, despite our previous observations that initially characterised ADAM-9 and -10 mRNA as being responsive to DHT. However, IGF-1 in synergy with DHT did significantly elevate mRNA levels ofboth ADAMs. In the case of ADAM-9 and -10 protein, the same trends of stimulation as found at the rnRNA level were shown by Western blot analysis when ADAM-9 and -10 signal intensity was normalised with the housekeeping protein ~-Tubulin. For EGF treatment, both ADAM-9 and -10 mRNA and protein levels were significantly elevated, and further investigation vm found this to be the case for each of these ADAMs proteins in the nuclear fractions of LNCaP cells. These studies are the first to describe extensively, the expression and hormonal/growth factor regulation of two members of the ADAMs family ( -9 and -1 0) in PCa. These observations imply that the expression of ADAM-9 and -10 have varied roles in PCa whilst it develops from androgen-sensitive (early stage disease), through to an androgeninsensitive (late-stage), metastatic disease. Further studies are now required to investigate the several key areas of focus that this research has revealed, including: • Investigation of the cellular mechanisms that are involved in actively transporting the ADAMs to the cell's nuclear compartment and the ADAMs functional roles in the cell nucleus. • The construction of a full-length human ADAM-10 mammalian expression construct with the introduction of a new Kozak sequence, that elevates ADAM-I 0 expression in an in vitro cell system are required, so that functional assays such as cell invasion, migration and attachment may be carried out to fmd the functional consequences of ADAM expression on cellular behaviour. • The regulation studies also need to be extended by confirming the preliminary observations that the nuclear levels of ADAMs may also be elevated by hormones and growth factors such as DHT, IGF-1 and EGF, as well as the regulation of levels of plasma membrany vesicle associated ADAM expression. Given the data presented in this study, it is likely the ADAMs have differential roles throughout the development of PCa due to their differential cellular localisation and synergistic growth-factor regulation. These observations, along with those further studies outlined above, are necessary in identifying these specific components ofPCa metastasis to which the ADAMs may contribute.

Load compensation for systems with non-stiff source using state feedback

Various load compensation schemes proposed in literature assume that voltage source at point of common coupling (PCC) is stiff. In practice, however, the load is remote from a distribution substation and is supplied by a feeder. In the presence of feeder impedance, the PWM inverter switchings distort both the PCC voltage and the source currents. In this paper load compensation with such a non-stiff source is considered. A switching control of the voltage source inverter (VSI) based on state feedback is used for load compensation with non-stiff source. The design of the state feedback controller requires careful considerations in choosing a gain matrix and in the generation of reference quantities. These aspects are considered in this paper. Detailed simulation and experimental results are given to support the control design.

A study of environmental and management drivers of non-co2 greenhouse gas emissions in Australian agro-ecosystems

Australian climate, soils and agricultural management practices are significantly different from those of the northern hemisphere nations. Consequently, experimental data on greenhouse gas production from European and North American agricultural soils and its interpretation are unlikely to be directly applicable to Australian systems.

Is it necessary to re-fuse a non-union of a hallux metatarsophalangeal joint arthrodesis?

BACKGROUND: The standard treatment for a non-union of the hallux metatarsophalangeal joint fusion has been to revise the fusion. Revision fusion is technically more demanding, often involving bone grafting, more substantial fixation and prolonged period of immobilization postoperatively. We present data to suggest that removal of hardware and debridement alone is an alternative treatment option. ---------- MATERIALS AND METHODS: A case note review identified patients with a symptomatic non-union after hallux metatarsophalangeal joint (MTPJ) fusion. It is our practice to offer these patients revision fusion or removal of hardware and debridement. For the seven patients that chose hardware removal and were left with a pseudarthrosis, a matched control group was selected from patients who had had successful fusions. Three outcome scores were used. Hallux valgus and dorsiflexion angles were recorded.---------- RESULTS: One hundred thirty-nine hallux MTPJ arthrodeses were carried out. Fourteen non-unions were identified. The rate of non-union in males and following previous hallux MTPJ surgery was 19% and 24%, respectively. In females undergoing a primary MTPJ fusion, the rate was 2.4%. Twelve non-union patients were reviewed at 27 months (mean). Eleven patients had elected to undergo removal of hardware and debridement. Four patients with pseudarthrosis were unhappy with the results and proceeded to either revision fusion or MTPJ replacement. Seven non-union patients, who had removal of hardware alone, had outcome scores marginally worse compared to those with successful fusions.---------- CONCLUSION: Removal of hardware alone is a reasonable option to offer as a relatively minor procedure following a failed arthrodesis of the first MTPJ. This must be accepted on the proviso that in this study four out of 11 (36%) patients proceeded to a revision first MTPJ fusion or first MTPJ replacement. We also found that the rate of non-union in primary first MTPJ fusion was significantly higher in males and those patients who had undergone previous surgery.

Testing for the non-separability of bi-ambiguous compounds

Separability is a concept that is very difficult to define, and yet much of our scientific method is implicitly based upon the assumption that systems can sensibly be reduced to a set of interacting components. This paper examines the notion of separability in the creation of bi-ambiguous compounds that is based upon the CHSH and CH inequalities. It reports results of an experiment showing that violations of the CHSH and CH inequality can occur in human conceptual combination.

Flow modeling in a novel non-perfusion conical bioreactor

We have developed a bioreactor vessel design which has the advantages of simplicity and ease of assembly and disassembly, and with the appropriately determined flow rate, even allows for a scaffold to be suspended freely regardless of its weight. This article reports our experimental and numerical investigations to evaluate the performance of a newly developed non-perfusion conical bioreactor by visualizing the flow through scaffolds with 45° and 90° fiber lay down patterns. The experiments were conducted at the Reynolds numbers (Re) 121, 170, and 218 based on the local velocity and width of scaffolds. The flow fields were captured using short-time exposures of 60 µm particles suspended in the bioreactor and illuminated using a thin laser sheet. The effects of scaffold fiber lay down pattern and Reynolds number were obtained and correspondingly compared to results obtained from a computational fluid dynamics (CFD) software package. The objectives of this article are twofold: to investigate the hypothesis that there may be an insufficient exchange of medium within the interior of the scaffold when using our non-perfusion bioreactor, and second, to compare the flows within and around scaffolds of 45° and 90° fiber lay down patterns. Scaffold porosity was also found to influence flow patterns. It was therefore shown that fluidic transport could be achieved within scaffolds with our bioreactor design, being a non-perfusion vessel. Fluid velocities were generally same of the same or one order lower in magnitude as compared to the inlet flow velocity. Additionally, the 90° fiber lay down pattern scaffold was found to allow for slightly higher fluid velocities within, as compared to the 45° fiber lay down pattern scaffold. This was due to the architecture and pore arrangement of the 90° fiber lay down pattern scaffold, which allows for fluid to flow directly through (channel-like flow).

Serious tax non compliance : motivation and guardianship

This article reviews what international evidence exists on the impact of civil and criminal sanctions upon serious tax noncompliance by individuals. This construct lacks sharp definitional boundaries but includes large tax fraud and large-scale evasion that are not dealt with as fraud. Although substantial research and theory have been developed on general tax evasion and compliance, their conclusions might not apply to large-scale intentional fraudsters. No scientifically defensible studies directly compared civil and criminal sanctions for tax fraud, although one U.S. study reported that significantly enhanced criminal sanctions have more effects than enhanced audit levels. Prosecution is public, whereas administrative penalties are confidential, and this fact encourages those caught to pay heavy penalties to avoid publicity, a criminal record, and imprisonment.

Reliability prediction using the non-parametric explicit hazard model : a case study

Survival probability prediction using covariate-based hazard approach is a known statistical methodology in engineering asset health management. We have previously reported the semi-parametric Explicit Hazard Model (EHM) which incorporates three types of information: population characteristics; condition indicators; and operating environment indicators for hazard prediction. This model assumes the baseline hazard has the form of the Weibull distribution. To avoid this assumption, this paper presents the non-parametric EHM which is a distribution-free covariate-based hazard model. In this paper, an application of the non-parametric EHM is demonstrated via a case study. In this case study, survival probabilities of a set of resistance elements using the non-parametric EHM are compared with the Weibull proportional hazard model and traditional Weibull model. The results show that the non-parametric EHM can effectively predict asset life using the condition indicator, operating environment indicator, and failure history.

Zoledronic acid once-yearly : what role in the prevention of non-vertebral osteoporotic fractures?

Osteoporosis is the most common bone disease. Low levels of oestrogens or testosterone are risk factors for primary osteoporosis. The most common cause of secondary osteoporosis is glucocorticoid treatment, but there are many other secondary causes of osteoporosis. Osteoporosis can be secondary to anti-oestrogen treatment for hormone-sensitive breast cancer and to androgen-deprivation therapy for prostate cancer. Zoledronic is the most potent bisphosphonate at inhibiting bone resorption. In osteoporosis, zoledronic acid increases bone mineral density for at least a year after a single intravenous administration. The efficacy and safety of extended release (once-yearly) zoledronic acid in the treatment of osteoporosis is reviewed.

Non-linear oscillations assessment of the distribution static compensator operating in voltage control mode

This article deals with the non-linear oscillations assessment of a distribution static comensator ooperating in voltage control mode using the bifurcation theory. A mathematical model of the distribution static compensator in the voltage control mode to carry out the bifurcation analysis is derived. The stabiity regions in the Thevein equivalent plane are computed. In addition, the stability regions in the control gains space, as well as the contour lines for different Floquet multipliers are computed. The AC and DC capacitor impacts on the stability are analyzed through the bifurcation theory. The observations are verified through simulaation studies. The computation of the stability region allows the assessment of the stable operating zones for a power system that includes a distribution static compensator operating in the voltage mode.

Predicting dry eye using non-invasive techniques of tear film surface assessment

PURPOSE. To measure tear film surface quality in healthy and dry eye subjects using three noninvasive techniques of tear film quality assessment and to establish the ability of these noninvasive techniques to predict dry eye. METHODS. Thirty four subjects participated in the study, and were classified as dry eye or normal, based on standard clinical assessments. Three non-invasive techniques were applied for measurement of tear film surface quality: dynamic-area high-speed videokeratoscopy (HSV), wavefront sensing (DWS) and lateral shearing interferometry (LSI). The measurements were performed in both natural blinking conditions (NBC) and in suppressed blinking conditions (SBC). RESULTS. In order to investigate the capability of each method to discriminate dry eye subjects from normal subjects, the receiver operating curve (ROC) was calculated and then the area under the curve (AUC) was extracted. The best result was obtained for the LSI technique (AUC=0.80 in SBC and AUC=0.73 in NBC), which was followed by HSV (AUC=0.72 in SBC and AUC=0.71 in NBC). The best result for DWS was AUC=0.64 obtained for changes in vertical coma in suppressed blinking conditions, while for normal blinking conditions the results were poorer. CONCLUSIONS. Non-invasive techniques of tear film surface assessment can be used for predicting dry eye and this can be achieved in natural blinking as well as suppressed blinking conditions. In this study, LSI showed the best detection performance, closely followed by the dynamic-area HSV. The wavefront sensing technique was less powerful, particularly in natural blinking conditions.

Multiple indicators of ambient and personal ultraviolet radiation exposure and risk of non-Hodgkin lymphoma (United States)

Recent epidemiologic studies have suggested that ultraviolet radiation (UV) may protect against non-Hodgkin lymphoma (NHL), but few, if any, have assessed multiple indicators of ambient and personal UV exposure. Using the US Radiologic Technologists study, we examined the association between NHL and self-reported time outdoors in summer, as well as average year-round and seasonal ambient exposures based on satellite estimates for different age periods, and sun susceptibility in participants who had responded to two questionnaires (1994–1998, 2003–2005) and who were cancer-free as of the earlier questionnaire. Using unconditional logistic regression, we estimated the odds ratio (OR) and 95% confidence intervals for 64,103 participants with 137 NHL cases. Self-reported time outdoors in summer was unrelated to risk. Lower risk was somewhat related to higher average year-round and winter ambient exposure for the period closest in time, and prior to, diagnosis (ages 20–39). Relative to 1.0 for the lowest quartile of average year-round ambient UV, the estimated OR for successively higher quartiles was 0.68 (0.42–1.10); 0.82 (0.52–1.29); and 0.64 (0.40–1.03), p-trend = 0.06), for this age period. The lower NHL risk associated with higher year-round average and winter ambient UV provides modest additional support for a protective relationship between UV and NHL.