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.

Twelve-month effectiveness of a parent-led, family-focused weight-management program for prepubertal children : a randomized, controlled trial

BACKGROUND Parenting-skills training may be an effective age-appropriate child behavior-modification strategy to assist parents in addressing childhood overweight. OBJECTIVE Our goal was to evaluate the relative effectiveness of parenting-skills training as a key strategy for the treatment of overweight children. DESIGN The design consisted of an assessor-blinded, randomized, controlled trial involving 111 (64% female) overweight, prepubertal children 6 to 9 years of age randomly assigned to parenting-skills training plus intensive lifestyle education, parenting-skills training alone, or a 12-month wait-listed control. Height, BMI, and waist-circumference z score and metabolic profile were assessed at baseline, 6 months, and 12 months (intention to treat). RESULTS After 12 months, the BMI z score was reduced by ∼10% with parenting-skills training plus intensive lifestyle education versus ∼5% with parenting-skills training alone or wait-listing for intervention. Waist-circumference z score fell over 12 months in both intervention groups but not in the control group. There was a significant gender effect, with greater reduction in BMI and waist-circumference z scores in boys compared with girls. CONCLUSION Parenting-skills training combined with promoting a healthy family lifestyle may be an effective approach to weight management in prepubertal children, particularly boys. Future studies should be powered to allow gender subanalysis.

Family-focused weight management program for five- to nine-year-olds incorporating parenting skills training with healthy lifestyle information to support behaviour modification

his case study aims to describe how general parenting principles can be used as part of parent-led, family-focused child weight management that is in line with current Australian Clinical Practice Guidelines. A parent-led, family-focused child weight management program was designed for use by dietitians with parents of young children (five- to nine-year-olds). The program utilises the cornerstones of overweight treatment: diet, activity, behaviour modification and family support delivered in an age-appropriate, family-focused manner. Parents participate in 16 sessions (4 parenting-focused, 8 lifestyle-focused and 4 individual telephone support calls) conducted weekly, fortnightly then monthly over six months. This case study illustrates how a family used the program, resulting in reduced degree of overweight and stabilised waist circumference in the child over 12 months. In conclusion, linking parenting skills to healthy family lifestyle education provides an innovative approach to family-focused child weight management. It addresses key Australian Clinical Practice Guidelines, works at the family level, and provides a means for dietitians to easily adopt age-appropriate behaviour modification as part of their practice.

An assessment of the potential of family day care as a nutrition promotion setting in South Australia

Articles > Journals > Health journals > Nutrition & Dietetics: The Journal of the Dieticians Association of Australia articles > March 2003 Article: An assessment of the potential of Family Day Care as a nutrition promotion setting in South Australia. (Original Research). Article from:Nutrition & Dietetics: The Journal of the Dieticians Association of Australia Article date:March 1, 2003 Author:Daniels, Lynne A.; Franco, Bunny; McWhinnie, Julie-Anne CopyrightCOPYRIGHT 2006 Dietitians Association of Australia. This material is published under license from the publisher through the Gale Group, Farmington Hills, Michigan. All inquiries regarding rights or concerns about this content should be directed to customer service. (Hide copyright information) Related articles Ads by Google TAFE Child Care Courses Government accredited courses. Study anytime, anywhere. www.seeklearning.com.au Get Work in Child Care Certificate III Children's Services 4 Day Course + Take Home Assessment HBAconsult.com.au Abstract Objective: To assess the potential role of Family Day Care in nutrition promotion for preschool children. Design and setting: A questionnaire to examine nutrition-related issues and practices was mailed to care providers registered in the southern region of Adelaide, South Australia. Care providers also supplied a descriptive, qualitative recall of the food provided by parents or themselves to each child less than five years of age in their care on the day closest to completion of the questionnaire. Subjects: 255 care providers. The response rate was 63% and covered 643 preschool children, mean 4.6 (SD 2.8) children per carer. Results: There was clear agreement that nutrition promotion was a relevant issue for Family Day Care providers. Nutrition and food hygiene knowledge was good but only 54% of respondents felt confident to address food quality issues with parents. Sixty-five percent of respondents reported non-neutral approaches to food refusal and dawdling (reward, punishment, cajoling) that overrode the child's control of the amount eaten. The food recalls indicated that most children (> 75%) were offered fruit at least once. Depending on the hours in care, (0 to 4, 5 to 8, greater than 8 hours), 20%, 32% and 55%, respectively, of children were offered milk and 65%, 82% and 87%, respectively, of children were offered high fat and sugar foods. Conclusions: Questionnaire responses suggest that many care providers are committed to and proactive in a range of nutrition promotion activities. There is scope for strengthening skills in the management of common problems, such as food refusal and dawdling, consistent with the current evidence for approaches to early feeding management that promote the development of healthy food preferences and eating patterns. Legitimising and empowering care providers in their nutrition promotion role requires clear policies, guide lines, adequate pre- and in-service training, suitable parent materials, and monitoring.

A new family of Marx generator based on resonant converter

This paper presents a novel topology to generate high voltage with utilization of slow and fast power switches. New concepts used in this topology include numbers of diode-capacitor units in parallel with resonant circuits which are connected to a positive buck-boost converter. The resonant circuit reverses the voltage polarity of the capacitors. This configuration has capability of generating a flexible high voltage with certain number of capacitors. The advantage of this topology is to use slow switches, less number of diodes and capacitors compare to Marx generator. Simulations have been performed to verify the proposed topology.

Vulnerability factors for disaster-induced child post-traumatic stress disorder : the case for low family resilience and previous mental illness

Objective: The aim of the present study was to investigate whether parent report of family resilience predicted children’s disaster-induced post-traumatic stress disorder (PTSD) and general emotional symptoms, independent of a broad range of variables including event-related factors, previous child mental illness and social connectedness. ---------- Methods: A total of 568 children (mean age = 10.2 years, SD = 1.3) who attended public primary schools, were screened 3 months after Cyclone Larry devastated the Innisfail region of North Queensland. Measures included parent report on the Family Resilience Measure and Strengths and Difficulties Questionnaire (SDQ)–emotional subscale and child report on the PTSD Reaction Index, measures of event exposure and social connectedness. ---------- Results: Sixty-four students (11.3%) were in the severe–very severe PTSD category and 53 families (28.6%) scored in the poor family resilience range. A lower family resilience score was associated with child emotional problems on the SDQ and longer duration of previous child mental health difficulties, but not disaster-induced child PTSD or child threat perception on either bivariate analysis, or as a main or moderator variable on multivariate analysis (main effect: adjusted odds ratio (ORadj) = 0.57, 95% confidence interval (CI) = 0.13–2.44). Similarly, previous mental illness was not a significant predictor of child PTSD in the multivariate model (ORadj = 0.75, 95%CI = 0.16–3.61). ---------- Conclusion: In this post-disaster sample children with existing mental health problems and those of low-resilience families were not at elevated risk of PTSD. The possibility that the aetiological model of disaster-induced child PTSD may differ from usual child and adolescent conceptualizations is discussed.

Infant and young child feeding indicators and determinants of poor feeding practices in India : secondary data analysis of National Family Health Survey 2005–06

Background: In India, poor feeding practices in early childhood contribute to the burden of malnutrition and infant and child mortality. Objective. To estimate infant and young child feeding indicators and determinants of selected feeding practices in India. Methods: The sample consisted of 20,108 children aged 0 to 23 months from the National Family Health Survey India 2005–06. Selected indicators were examined against a set of variables using univariate and multivariate analyses. Results: Only 23.5% of mothers initiated breastfeeding within the first hour after birth, 99.2% had ever breastfed their infant, 89.8% were currently breastfeeding, and 14.8% were currently bottle-feeding. Among infants under 6 months of age, 46.4% were exclusively breastfed, and 56.7% of those aged 6 to 9 months received complementary foods. The risk factors for not exclusively breastfeeding were higher household wealth index quintiles (OR for richest = 2.03), delivery in a health facility (OR = 1.35), and living in the Northern region. Higher numbers of antenatal care visits were associated with increased rates of exclusive breastfeeding (OR for ≥ 7 antenatal visits = 0.58). The rates of timely initiation of breastfeeding were higher among women who were better educated (OR for secondary education or above = 0.79), were working (OR = 0.79), made more antenatal clinic visits (OR for ≥ 7 antenatal visits = 0.48), and were exposed to the radio (OR = 0.76). The rates were lower in women who were delivered by cesarean section (OR = 2.52). The risk factors for bottle-feeding included cesarean delivery (OR = 1.44), higher household wealth index quintiles (OR = 3.06), working by the mother (OR=1.29), higher maternal education level (OR=1.32), urban residence (OR=1.46), and absence of postnatal examination (OR=1.24). The rates of timely complementary feeding were higher for mothers who had more antenatal visits (OR=0.57), and for those who watched television (OR=0.75). Conclusions: Revitalization of the Baby Friendly Hospital Initiative in health facilities is recommended. Targeted interventions may be necessary to improve infant feeding practices in mothers who reside in urban areas, are more educated, and are from wealthier households.

Pet food from bovine origin drying by green heat pump technology and fluidization

Bovine intestine was dried in a heat pump fluid bed combination. Minimum fluidisation velocity was calculated by Ergun Equation and some relations were established.

ACE research briefing paper 001 : the role of family in new business start-ups

This report uses data from the first two years of the CAUSEE study and focuses on the role of family in new business start-ups. While CAUSEE was not designed specifically to probe deeply into family matters the study does reveal interesting information on family orientated aspects including parental role models, family involvement on the start-up team, and family as a source of funding and advice. These findings can also be related to other information gathered as part of the comprehensive phone interviews that are used to gather the research data for CAUSEE. Furthermore we are also able to compare firm founders and 'Regular' nascent firm and young firm start-ups with their 'High Potential' counterparts in terms of their degrees of family involvement. Unless otherwise stated any differences or effects we comment on are 'statistically significant' at the five per cent level - that is, they are likely to reflect true differences or effects in the entire population of Australian start-ups.

Green roofs : understanding their benefits for Australia

Summary of Actions Towards Sustainable Outcomes Environmental Issues / Principal Impacts The increased growth of cities is intensifying its impact on people and the environment through: • increased use of energy for the heating and cooling of more buildings, leading to urban heat islands and more greenhouse gas emissions • increased amount of hard surfaces contributing to higher temperatures in cities and more stormwater runoff • degraded air quality and noise impact • reduced urban biodiversity • compromised health and general well-being of people Basic Strategies In many design situations boundaries and constraints limit the application of cutting EDGe actions. In these circumstances designers should at least consider the following: • Consider green roofs early in the design process in consultation with all stakeholders to enable maximised integration with building systems and to mitigate building cost (avoid constructing as a retrofit). • Design of the green roof as part of a building’s structural, mechanical and hydraulic systems could lead to structural efficiency, the ability to optimise cooling benefits and better integrated water recycling systems. • Inform the selection of the type of green roof by considering its function, for example designing for social activity, required maintenance/access regime, recycling of water or habitat regeneration or a combination of uses. • Evaluate existing surroundings to determine possible links to the natural environment and choice of vegetation for the green roof with availability of local plant supply and expertise. Cutting EDGe Strategies • Create green roofs to contribute positively to the environment through reduced urban heat island effect and building temperatures, to improved stormwater quality, increased natural habitats, provision of social spaces and opportunity for increased local food supply. • Maximise solar panel efficiency by incorporating with design of green roof. • Integrate multiple functions for a single green roof such as grey water recycling, food production, more bio-diverse plantings, air quality improvement and provision of delightful spaces for social interaction. Synergies & references • BEDP Environment Design Guide DES 53: Roof and Facade Gardens GEN 4: Positive Development – designing for Net Positive Impacts TEC 26: Living Walls - a way to green the built environment • Green Roofs Australia: www.greenroofs.wordpress.com • International Green Roof Association: www.igra-world.com • Green Roofs for Healthy Cities (USA): www.greenroofs.org • Centre for Urban Greenery and Ecology (Singapore): http://research.cuge.com.sg