The mechanism of maturation of insulin-like growth factor-1

This study, to elucidate the role of des(1-3)IGF-I in the maturation of IGF-I,used two strategies. The first was to detect the presence of enzymes in tissues, which would act on IGF-I to produce des(1-3)IGF-I, and the second was to detect the potential products of such enzymic activity, namely Gly-Pro-Glu(GPE), Gly-Pro(GP) and des(l- 3)IGF-I. No neutral tripeptidyl peptidase (TPP II), which would release the tripeptide GPE from IGF-I, was detected in brain, urine nor in red or white blood cells. The TPPlike activity which was detected, was attributed to a combined action of a dipeptidyl peptidase (DPP N) and an aminopeptidase (AP A). A true TPP II was, however, detected in platelets. Two purified TPP II enzymes were investigated but they did not release GPE from IGF-I under a variety of conditions. Consequently, TPP II seemed unlikely to participate in the formation of des(1-3)IGF-I. In contrast, an acidic tripeptidyl peptidase activity (TPP I) was detected in brain and colostrum, the former with a pH optimum of 4.5 and the latter 3.8. It seems likely that such an enzyme would participate in the formation of des( 1-3 )IGF-I in these tissues in vitro, ie. that des(1-3)IGF-I may have been produced as an artifact in the isolation of IGF-I from brain and colostrum in acidic conditions. This contrasts with suggestions of an in vivo role for des(1-3)IGF-I, as reported by others. The activity of a dipeptidyl peptidase N (DPP N) from urine, which should release the dipeptide GP from IGF-I, was assessed under a variety of conditions and with a variety of additives and potential enzyme stimulants, but there was no release of GP. The DPP N also exhibited a transferase activity with synthetic substrates in the presence of dipeptides, at lower concentrations than previously reported for other acceptors or other proteolytic enzymes. In addition, a low concentration of a product,possibly the tetrapeptide Gly-Pro-Gly-Leu, was detected with the action of the enzyme on IGF-I in the presence of the dipeptide Gly-Leu. As part of attempts to detect tissue production of des(1-3)IGF-I, a monoclonal antibody (MAb ), directed towards the GPE- end ofiGF-I was produced by immunisation with a 10-mer covalently attached to a carrier protein. By the use of indirect ELISA and inhibitor studies, the MAb was shown to selectively recognise peptides with anNterminal GPE- sequence, and applied to the indirect detection of des(1-3)IGF-I. The concentration of GPE in brain, measured by mass spectrometry ( MS), was low, and the concentration of total IGF-I (measured by ELISA with a commercial polyclonal antibody [P Ab]) was 40 times higher at 50 nmol/kg. This also, was not consistent with the action of a tripeptidyl peptidase in brain that converted all IGF-I to des(1-3)IGF-I plus GPE. Contrasting ELISA results, using the MAb prepared in this study, suggest an even higher concentration of intact IGF-I of 150 nmollkg. This would argue against the presence of any des( 1-3 )IGF-I in brain, but in turn, this indicates either the presence of other substances containing a GPE amino-terminus or other cross reacting epitope. Although the results of the specificity studies reported in Chapter 5 would make this latter possibility seem unlikely, it cannot be completely excluded. No GP was detected in brain by MS. No GPE was detected in colostrum by capillary electrophoresis (CE) but the interference from extraneous substances reduced the detectability of GPE by CE and this approach would require further, prior, purification and concentration steps. A molecule, with a migration time equal to that of the peptide GP, was detected in colostrum by CE, but the concentration (~ 10 11mo/L) was much higher than the IGF-I concentration measured by radio-immunoassay using a PAb (80 nmol/L) or using a Mab (300-400 nmolL). A DPP IV enzyme was detected in colostrum and this could account for the GP, derived from substrates other than IGF-1. Based on the differential results of the two antibody assays, there was no indication of the presence of des(1-3)IGF-I in brain or colostrum. In the absence of any enzyme activity directed towards the amino terminus of IGF-I and the absence any potential products, IGF-I, therefore, does not appear to "mature" via des(1-3)IGF-I in the brain, nor in the neutral colostrum. In spite of these results which indicate the absence of an enzymic attack on IGF-I and the absence of the expected products in tissues, the possibility that the conversion of IGF-I may occur in neutral conditions in limited amounts, cannot be ruled out. It remains possible that in the extracellular environment of the membrane, a complex interaction of IGF-I, binding protein, aminopeptidase(s) and receptor, produces des(1- 3)IGF-I as a transient product which is bound to the receptor and internalised.

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.

Advanced Gen-1, 2 and 3 biofuels research in Australia - Fuelling advanced biofuels training for international scientists

Flinders University and Queensland University of Technology, biofuels research interests cover a broad range of activities. Both institutions are seeking to overcome the twin evils of "peak oil" (Hubbert 1949 & 1956) and "global warming" (IPPC 2007, Stern 2006, Alison 2010), through development of Generation 1, 2 and 3 (Gen-1, 2 & 3) biofuels (Clarke 2008, Clarke 2010). This includes development of parallel Chemical Biorefinery, value-added, co-product chemical technologies, which can underpin the commercial viability of the biofuel industry. Whilst there is a focused effort to develop Gen-2 & 3 biofuels, thus avoiding the socially unacceptable use of food based Gen-1 biofuels, it must also be recognized that as yet, no country in the world has produced sustainable Gen-2 & 3 biofuel on a commercial basis. For example, in 2008 the United States used 38 billion litres (3.5% of total fuel use) of Gen-1 biofuel; in 2009/2010 this will be 47.5 billion litres (4.5% of fuel use) and in 2018 this has been estimated to rise to 96 billion litres (9% of total US fuel use). Brazil in 2008 produced 24.5 billion litres of ethanol, representing 37.3% of the world’s ethanol use for fuel and Europe, in 2008, produced 11.7 billion litres of biofuel (primarily as biodiesel). Compare this to Australia’s miserly biofuel production in 2008/2009 of 180 million litres of ethanol and 75 million litres of biodiesel, which is 0.4% of our fuel consumption! (Clarke, Graiver and Habibie 2010) To assist in the development of better biofuels technologies in the Asian developing regions the Australian Government recently awarded the Materials & BioEnergy Group from Flinders University, in partnership with the Queensland University of Technology, an Australian Leadership Award (ALA) Biofuel Fellowship program to train scientists from Indonesia and India about all facets of advanced biofuel technology.

A ∑GIi/D/1/∞ queue with heterogeneous input/output slot times

In this paper, we present a ∑GIi/D/1/∞ queue with heterogeneous input/output slot times. This queueing model can be regarded as an extension of the ordinary GI/D/1/∞ model. For this ∑GIi/D/1/∞ queue, we assume that several input streams arrive at the system according to different slot times. In other words, there are different slot times for different input/output processes in the queueing model. The queueing model can therefore be used for an ATM multiplexer with heterogeneous input/output link capacities. Several cases of the queueing model are discussed to reflect different relationships among the input/output link capacities of an ATM multiplexer. In the queueing analysis, two approaches: the Markov model and the probability generating function technique, are adopted to develop the queue length distributions observed at different epochs. This model is particularly useful in the performance analysis of ATM multiplexers with heterogeneous input/output link capacities.

Old Brisbane Botanic Gardens: Conservation Plan Review 2005, Report of Stage 1 Heritage Significance and Conservation Policies

Report for City Design, for Environment and Parks, within the Brisbane City Council. Context of this Project A Conservation Study for the Old Brisbane Botanic Gardens, formerly called the Brisbane City Botanic Gardens, was finalised in 1995 and prepared by Jeannie Sim for the Landscape Section of Brisbane City Council, the same author of the present report. This unpublished report was the first conservation plan prepared for the place and it was recommended that it be reviewed in five years time. That time has arrived finally with the preparation of the 2005 Review. The present project was commissioned by City Design on behalf of Environment and Parks Section of Brisbane City Council. The author has purposely chosen to call the study site the 'Old Brisbane Botanic Gardens' (OBBG) to differentiate it from the Brisbane Botanic Gardens, Mt. Coot-tha (BBG-MC), and to maintain the claim for this original garden to remain as a botanic garden for Brisbane. This name immediately brings to mind an association with history, as in the precedent set by the naming of the nearby 'Old Government House' at Gardens Point.

Three-dimensional hydrogen-bonded structures in the guanidinium salts of the monocyclic dicarboxylic acids rac-trans-cyclohexane-1,2-dicarboxylic acid (2:1, anhydrous), isophthalic acid (1:1, monohydrate) and terephthalic acid (2:1, trihydrate).

The structures of bis(guanidinium)rac-trans-cyclohexane-1,2-dicarboxylate, 2(CH6N3+) C8H10O4- (I), guanidinium 3-carboxybenzoate monohydrate CH6N3+ C8H5O4- . H2O (II) and bis(guanidinium) benzene-1,4-dicarboxylate trihydrate, 2(CH6N3+) C8H4O4^2- . 3H2O (III) have been determined and the hydrogen bonding in each examined. All three compounds form three-dimensional hydrogen-bonded framework structures. In anhydrous (I), both guanidinium cations give classic cyclic R2/2(8) N--H...O,O'(carboxyl) and asymmetric cyclic R1/2(6) hydrogen-bonding interactions while one cation gives an unusual enlarged cyclic interaction with O acceptors of separate ortho-related carboxyl groups [graph set R2/2(11)]. Cations and anions also associate across inversion centres giving cyclic R2/4(8) motifs. In the 1:1 guanidinium salt (II), the cation gives two separate cyclic R1/2(6) interactions, one with a carboxyl O-acceptor, the other with the water molecule of solvation. The structure is unusual in that both carboxyl groups give short inter-anion O...H...O contacts, one across a crystallographic inversion centre [2.483(2)\%A], the other about a two-fold axis of rotation [2.462(2)\%A] with a half-occupancy hydrogen delocalized on the symmetry element in each. The water molecule links the cation--anion ribbon structures into a three-dimensional framework. In (III), the repeating molecular unit comprises a benzene-1,4-dicarboxylate dianion which lies across a crystallographic inversion centre, two guanidinium cations and two water molecules of solvation (each set related by two-fold rotational symmetry), and a single water molecule which lies on a two-fold axis. Each guanidinium cation gives three types of cyclic interactions with the dianions: one R^1^~2~(6), the others R2/3(8) and R3/3(10) (both of these involving the water molecules), giving a three-dimensional structure through bridges down the b cell direction. The water molecule at the general site also forms an unusual cyclic R2/2(4) homodimeric association across an inversion centre [O--H...O, 2.875(2)\%A]. The work described here provides further examples of the common cyclic guanidinium cation...carboxylate anion hydrogen-bonding associations as well as featuring other less common cyclic motifs.