p53 prevents progression of nevi to melanoma predominantly through cell cycle regulation

p53 is the central member of a critical tumor suppressor pathway in virtually all tumor types, where it is silenced mainly by missense mutations. In melanoma, p53 predominantly remains wild type, thus its role has been neglected. To study the effect of p53 on melanocyte function and melanomagenesis, we crossed the 'high-p53'Mdm4+/- mouse to the well-established TP-ras0/+ murine melanoma progression model. After treatment with the carcinogen dimethylbenzanthracene (DMBA), TP-ras0/+ mice on the Mdm4+/- background developed fewer tumors with a delay in the age of onset of melanomas compared to TP-ras0/+ mice. Furthermore, we observed a dramatic decrease in tumor growth, lack of metastasis with increased survival of TP-ras0/+: Mdm4+/- mice. Thus, p53 effectively prevented the conversion of small benign tumors to malignant and metastatic melanoma. p53 activation in cultured primary melanocyte and melanoma cell lines using Nutlin-3, a specific Mdm2 antagonist, supported these findings. Moreover, global gene expression and network analysis of Nutlin-3-treated primary human melanocytes indicated that cell cycle regulation through the p21WAF1/CIP1 signaling network may be the key anti-melanomagenic activity of p53.

Comparison between electrically evoked and voluntary isometric contractions for biceps brachii muscle oxidative metabolism using near-infrared spectroscopy

This study compared voluntary (VOL) and electrically evoked isometric contractions by muscle stimulation (EMS) for changes in biceps brachii muscle oxygenation (tissue oxygenation index, ΔTOI) and total haemoglobin concentration (ΔtHb = oxygenated haemoglobin + deoxygenated haemoglobin) determined by near-infrared spectroscopy. Twelve men performed EMS with one arm followed 24 h later by VOL with the contralateral arm, consisting of 30 repeated (1-s contraction, 1-s relaxation) isometric contractions at 30% of maximal voluntary contraction (MVC) for the first 60 s, and maximal intensity contractions thereafter (MVC for VOL and maximal tolerable current at 30 Hz for EMS) until MVC decreased ∼30% of pre-exercise MVC. During the 30 contractions at 30% MVC, ΔTOI decrease was significantly (P < 0.05) greater and ∼tHb was significantly (P < 0.05) lower for EMS than VOL, suggesting that the metabolic demand for oxygen in EMS is greater than VOL at the same torque level. However, during maximal intensity contractions, although EMS torque (∼40% of VOL) was significantly (P < 0.05) lower than VOL, ΔTOI was similar and ΔtHb was significantly (P < 0.05) lower for EMS than VOL towards the end, without significant differences between the two sessions in the recovery period. It is concluded that the oxygen demand of the activated biceps brachii muscle in EMS is comparable to VOL at maximal intensity. © Springer-Verlag 2009.

Neuropsychological development in children with early and continuously treated phenylketonuria : association with biochemical markers

PKU is a genetically inherited inborn error of metabolism caused by a deficiency of the enzyme phenylalanine hydroxylase. The failure of this enzyme causes incomplete metabolism of protein ingested in the diet, specifically the conversion of one amino acid, phenylalanine, to tyrosine, which is a precursor to the neurotransmitter dopamine. Rising levels of phenylalanine is toxic to the developing brain, disrupting the formation of white matter tracts. The impact of tyrosine deficiency is not as well understood, but is hypothesized to lead to a low dopamine environment for the developing brain. Detection in the newborn period and continuous treatment (a low protein phe-restricted diet supplemented with phenylalanine-free protein formulas) has resulted in children with early and continuously treated PKU now developing normal I.Q. However, deficits in executive function (EF) are common, leading to a rate of Attention Deficit Hyperactivity Disorder (ADHD) up to five times the norm. EF worsens with exposure to higher phenylalanine levels, however recent research has demonstrated that a high phenylalanine to tyrosine ratio (phenylalanine:tyrosine ratio), which is hypothesised to lead to poorer dopamine function, has a more negative impact on EF than phenylalanine levels alone. Research and treatment of PKU is currently phenylalanine-focused, with little investigation of the impact of tyrosine on neuropsychological development. There is no current consensus as to the veracity of tyrosine monitoring or treatment in this population. Further, the research agenda in this population has demonstrated a primary focus on EF impairment alone, even though there may be additional neuropsychological skills compromised (e.g., mood, visuospatial deficits). The aim of this PhD research was to identify residual neuropsychological deficits in a cohort of children with early and continuously treated phenylketonuria, at two time points in development (early childhood and early adolescence), separated by eight years. In addition, this research sought to determine which biochemical markers were associated with neuropsychological impairments. A clinical practice survey was also undertaken to ascertain the current level of monitoring/treatment of tyrosine in this population. Thirteen children with early and continuously treated PKU were tested at mean age 5.9 years and again at mean age 13.95 years on several neuropsychological measures. Four children with hyperphenylalaninemia (a milder version of PKU) were also tested at both time points and provide a comparison group in analyses. Associations between neuropsychological function and biochemical markers were analysed. A between groups analysis in adolescence was also conducted (children with PKU compared to their siblings) on parent report measures of EF and mood. Minor EF impairments were evident in the PKU group by age 6 years and these persisted into adolescence. Life-long exposure to high phenylalanine:tyrosine ratio and/or low tyrosine independent of phenylalanine were significantly associated with EF impairments at both time points. Over half the children with PKU showed severe impairment on a visuospatial task, and this was associated only with concurrent levels of tyrosine in adolescence. Children with PKU also showed a statistically significant decline in a language comprehension task from 6 years to adolescence (going from normal to subnormal), this deficit was associated with lifetime levels of phenylalanine. In comparison, the four children with hyperphenylalaninemia demonstrated normal function at both time points, across all measures. No statistically significant differences were detected between children with PKU and their siblings on the parent report of EF and mood. However, depressive symptoms were significantly correlated with: EF; long term high phe:tyr exposure; and low tyrosine levels independent of phenylalanine. The practice survey of metabolic clinics from 12 countries indicated a high level of variability in terms of monitoring/treatment of tyrosine in this population. Whilst over 80% of clinics surveyed routinely monitored tyrosine levels in their child patients, 25% reported treatment strategies to increase tyrosine (and thereby lower the phenylalanine:tyrosine ratio) under a variety of patient presentation conditions. Overall, these studies have shown that EF impairments associated with PKU provide support for the dopamine-deficiency model. A language comprehension task showed a different trajectory, serving a timely reminder that non-EF functions also remain vulnerable in this population; and that normal function in childhood does not guarantee normal function by adolescence. Mood impairments were associated with EF impairments as well as long term measures of phenylalanine:tyrosine and/or tyrosine. The implications of this research for enhanced clinical guidelines are discussed given varied current practice.

Water sensitive urban design through the lens of urban metabolism

Stormwater pollution has been recognised as one of the main causes of aquatic ecosystem degradation and poses a significant threat to both the goal of ecological sustainable development as well as human health and wellbeing. In response, water sensitive urban design (WSUD) practices have been put forward as a strategy to mitigate the detrimental impacts of urban stormwater runoff quality and to safeguard ecosystem functions. However, despite studies that support its efficiency in urban stormwater management, the mainstreaming of WSUD remains a significant challenge. This paper proposes that viewing WSUD through the lens of the integrated urban metabolism framework which encourages an interdisciplinary approach and facilitates dialogue through knowledge transfer is a strategy in which the implementation of WSUD can be mainstreamed.

Sustainability and urban settlements: urban metabolism as a framework for achieving sustainable development

Urban settlements, with their role as economic and governance nerve centres, are rapidly expanding in size and in consumption of resources, and consequently have significant impacts on the environment. The transition to an ‘eco-city’ - an urban settlement that adopts the goals and principles in the urban metabolism model - needs to occur to meet the challenges posed by a multitude of pressures including population growth, climate change and resource depletion. Thus, the adoption and integration of ‘sustainable development’ into the management of urban growth is one of the most critical governance issues for urban settlements. A framework in which sustainable development can be achieved is through the lenses of the established theoretical concept of ‘urban metabolism’. The key facet of the proposed ‘Integrated Urban Metabolism Framework’ is the provision of a platform whereby different fields can appreciate, absorb and learn from other areas, to increase the understanding of where each and every one of the pieces fit together in order to create a larger, holistic approach to the currently stagnant problem of unsustainable development.

AtBAG7, an Arabidopsis Bcl-2-associated athanogene, resides in the endoplasmic reticulum and is involved in the unfolded protein response

The Bcl-2-associated athanogene (BAG) family is an evolutionarily conserved, multifunctional group of cochaperones that perform diverse cellular functions ranging from proliferation to growth arrest and cell death in yeast, in mammals, and, as recently observed, in plants. The Arabidopsis genome contains seven homologs of the BAG family, including four with domain organization similar to animal BAGs. In the present study we show that an Arabidopsis BAG, AtBAG7, is a uniquely localized endoplasmic reticulum (ER) BAG that is necessary for the proper maintenance of the unfolded protein response (UPR). AtBAG7was shown to interact directly in vivo with themolecular chaperone, AtBiP2, by bimolecular fluorescence complementation assays, and the interaction was confirmed by yeast two-hybrid assay. Treatment with an inducer of UPR, tunicamycin, resulted in accelerated cell death of AtBAG7-null mutants. Furthermore, AtBAG7 knockouts were sensitive to known ER stress stimuli, heat and cold. In these knockouts heat sensitivity was reverted successfully to the wild-type phenotype with the addition of the chemical chaperone, tauroursodexycholic acid (TUDCA). Real-time PCR of ER stress proteins indicated that the expression of the heat-shock protein, AtBiP3, is selectively up-regulated in AtBAG7-null mutants upon heat and cold stress. Our results reveal an unexpected diversity of the plant's BAG gene family and suggest that AtBAG7 is an essential component of the UPR during heat and cold tolerance, thus confirming the cytoprotective role of plant BAGs.

Evaluation of novel Streptococcus pyogenes vaccine candidates incorporating multiple conserved sequences from the C-repeat region of the M-protein

A major challenge for Streptococcus pyogenes vaccine development is the identification of epitopes that confer protection from infection by multiple S. pyogenes M-types. Here we have identified and characterised the distribution of common variant sequences from individual repeat units of the C-repeat region (CRR) of M-proteins representing 77 different M-types. Three polyvalent fusion vaccine candidates (SV1, SV2 and SV3) incorporating the most common variants were subsequently expressed and purified, and demonstrated to be alpha-helical by Circular Dichroism (CD), a secondary conformational characteristic of the CRR in the M-protein. Antibodies raised against each of these constructs recognise M-proteins that vary in their CRR, and bind to the surface of multiple S. pyogenes isolates. Antibodies raised against SV1, containing five variant sequences, also kill heterologous S. pyogenes isolates in in vitro bactericidal assays. Further structural characterisation of this construct demonstrated the conformation of SV1 was stable at different pHs, and thermal unfolding of SV1 a reversible process. Our findings demonstrate that linkage of multiple variant sequences into a single recombinant construct overcomes the need to embed the variant sequences in foreign helix promoting flanking sequences for conformational stability, and demonstrates the viability of the polyvalent candidates as global S. pyogenes vaccine candidates.

Development and validation of an expedited 10g protein counter (EP-10) for dietary protein intake quantification

Precise protein quantification is essential in clinical dietetics, particularly in the management of renal, burn and malnourished patients. The EP-10 was developed to expedite the estimation of dietary protein for nutritional assessment and recommendation. The main objective of this study was to compare the validity and efficacy of the EP-10 with the American Dietetic Association’s “Exchange List for Meal Planning” (ADA-7g) in quantifying dietary protein intake, against computerised nutrient analysis (CNA). Protein intake of 197 food records kept by healthy adult subjects in Singapore was determined thrice using three different methods – (1) EP-10, (2) ADA-7g and (3) CNA using SERVE program (Version 4.0). Assessments using the EP-10 and ADA-7g were performed by two assessors in a blind crossover manner while a third assessor performed the CNA. All assessors were blind to each other’s results. Time taken to assess a subsample (n=165) using the EP-10 and ADA-7g was also recorded. Mean difference in protein intake quantification when compared to the CNA was statistically non-significant for the EP-10 (1.4 ± 16.3 g, P = .239) and statistically significant for the ADA-7g (-2.2 ± 15.6 g, P = .046). Both the EP-10 and ADA-7g had clinically acceptable agreement with the CNA as determined via Bland-Altman plots, although it was found that EP-10 had a tendency to overestimate with protein intakes above 150 g. The EP-10 required significantly less time for protein intake quantification than the ADA-7g (mean time of 65 ± 36 seconds vs. 111 ± 40 seconds, P < .001). The EP-10 and ADA-7g are valid clinical tools for protein intake quantification in an Asian context, with EP-10 being more time efficient. However, a dietician’s discretion is needed when the EP-10 is used on protein intakes above 150g.

A longitudinal assessment of chronic wound fluid to detect biochemical indicators of healing

Chronic venous leg ulcers are a detrimental health issue plaguing our society, resulting in long term pain, immobility and decreased quality of life for a large proportion of sufferers. The frequency of these chronic wounds has led current research to focus on the wound environment to provide important information regarding the prolonged, fluctuated or static healing patterns of these wounds. Disruption to the normal wound healing process results in release of multiple factors in the wound environment that could correlate to wound chronicity. These biochemical factors can often be detected through non-invasively sampling chronic wound fluid (CWF) from the site of injury. Of note, whilst there are numerous studies comparing acute and chronic wound fluids, there have not been any reports in the literature employing a longitudinal study in order to track biochemical changes in wound fluid as patients transition from a non-healing to healed state. Initially the objective of this study was to identify biochemical changes in CWF associated with wound healing using a proteomic approach. The proteomic approach incorporated a multi-dimensional liquid chromatography fractionation technique coupled with mass spectrometry (MS) to enable identification of proteins present in lower concentrations in CWF. Not surprisingly, many of the proteins identified in wound fluid were acute phase proteins normally expressed during the inflammatory phase of healing. However, the number of proteins positively identified by MS was quite low. This was attributed to the diverse range in concentration of protein species in CWF making it challenging to detect the diagnostically relevant low molecular weight proteins. In view of this, SELDI-TOF MS was also explored as a means to target low molecular weight proteins in sequential patient CWF samples during the course of healing. Unfortunately, the results generated did not yield any peaks of interest that were altered as wounds transitioned to a healed state. During the course of proteomic assessment of CWF, it became evident that a fraction of non-proteinaceous compounds strongly absorbed at 280 nm. Subsequent analyses confirmed that most of these compounds were in fact part of the purine catabolic pathway, possessing distinctive aromatic rings and which results in high absorbance at 254 nm. The accumulation of these purinogenic compounds in CWF suggests that the wound bed is poorly oxygenated resulting in a switch to anaerobic metabolism and consequently ATP breakdown. In addition, the presence of the terminal purine catabolite, uric acid (UA), indicates that the enzyme xanthine oxidoreductase (XOR) catalyses the reaction of hypoxanthine to xanthine and finally to UA. More importantly, the studies provide evidence for the first time of the exogenous presence of XOR in CWF. XOR is the only enzyme in humans capable of catalysing the production of UA in conjunction with a burst of the highly reactive superoxide radical and other oxidants like H2O2. Excessive release of these free radicals in the wound environment can cause cellular damage disrupting the normal wound healing process. In view of this, a sensitive and specific assay was established for monitoring low concentrations of these catabolites in CWF. This procedure involved combining high performance liquid chromatography (HPLC) with tandem mass spectrometry and multiple reaction monitoring (MRM). This application was selective, using specific MRM transitions and HPLC separations for each analyte, making it ideal for the detection and quantitation of purine catabolites in CWF. The results demonstrated that elevated levels of UA were detected in wound fluid obtained from patients with clinically worse ulcers. This suggests that XOR is active in the wound site generating significant amounts of reactive oxygen species (ROS). In addition, analysis of the amount of purine precursors in wound fluid revealed elevated levels of purine precursors in wound fluid from patients with less severe ulcers. Taken together, the results generated in this thesis suggest that monitoring changes of purine catabolites in CWF is likely to provide valuable information regarding the healing patterns of chronic venous leg ulcers. XOR catalysis of purine precursors not only provides a method for monitoring the onset, prognosis and progress of chronic venous leg ulcers, but also provides a potential therapeutic target by inhibiting XOR, thus blocking UA and ROS production. Targeting a combination of these purinogenic compounds and XOR could lead to the development of novel point of care diagnostic tests. Therefore, further investigation of these processes during wound healing will be worthwhile and may assist in elucidating the pathogenesis of this disease state, which in turn may lead to the development of new diagnostics and therapies that target these processes.

The role of insulin and IGF2 signalling on metabolic pathways in prostate cancer progression

Prostate cancer (CaP) is the most commonly diagnosed cancer in males in Australia, North America, and Europe. If found early and locally confined, CaP can be treated with radical prostatectomy or radiation therapy; however, 25-40% patients will relapse and go on to advanced disease. The most common therapy in these cases is androgen deprivation therapy (ADT), which suppresses androgen production from the testis. Lack of the testicular androgen supply causes cells of the prostate to undergo apoptosis. However, in some cases the regression initially seen with ADT eventually gives way to a growth of a population of cancerous cells that no longer require testicular androgens. This phenotype is essentially fatal and is termed castrate resistant prostate cancer (CRPC). In addition to eventual regression, there are many undesirable side effects which accompany ADT, including development of a metabolic syndrome, which is defined by the U.S. National Library of Medicine as “a combination of medical disorders that increase the risk of developing cardiovascular disease and diabetes.” This project will focus on the effect of ADT induced hyperinsulinemia, as mimicked by treating androgen receptor positive CaP cells with insulin in a serum (hormone) deprived environment. While this side effect is not widely explored, in this thesis it is demonstrated for the first time that insulin upregulates pathways important to CaP progression. Our group has previously shown that during CaP progression, the enzymes necessary for de novo steroidogenesis are upregulated in the LNCaP xenograft model, total steroid levels are increased in tumours compared to pre castrate levels, and de novo steroidogenesis from radio-labelled acetate has been demonstrated. Because of the CaP dependence on AR for survival, we and other groups believe that CaP cells carry out de novo steroidogenesis to survive in androgen deprived conditions. Because (a) men on ADT often develop metabolic syndrome, and (b) men with lifestyle-induced obesity and hyperinsulinemia have worse prognosis and faster disease progression, and because (c) insulin causes steroidogenesis in other cell lines, the hypothesis that insulin may contribute to CaP progression through upregulation of steroidogenesis was explored. Insulin upregulates steroidogenesis enzymes at the mRNA level in three AR positive cell lines, as well as upregulating these enzymes at the protein level in two cell lines. It has also been demonstrated that insulin increases mitochondrial (functional) levels of steroid acute regulatory protein (StAR). Furthermore, insulin causes increased levels of total steroids in and induction of de novo steroid synthesis by insulin has been demonstrated at levels induced sufficient to activate AR. The effect of insulin analogs on CaP steroidogenesis in LNCaP and VCaP cells has also been investigated because epidemiological studies suggest that some of the analogs developed may have more cancer stimulatory effects than normal insulin. In this project, despite the signalling differences between glargine, X10, and insulin, these analogs did not appear to induce steroidogenesis any more potently that normal insulin. The effect of insulin of MCF7breast cancer cells was also investigated with results suggesting that breast cancer cells may be capable of de novo steroidogenesis, and that increase in estradiol production may be exacerbated by insulin. Insulin has also been long known to stimulate lipogenesis in the liver and adipocytes, and has been demonstrated to increase lipogenesis in breast cancer cells; therefore, investigation of the effect of insulin on lipogenesis, which is a hallmark of aggressive cancers, was investigated. In CaP progression sterol regulatory element binding protein (SREBP) is dysregulated and upregulates fatty acid synthase (FASN), acetyl CoA-carboxylase, and other lipogenesis genes. SREBP is important for steroidogenesis and in this project has been shown to be upregulated by insulin in CaP cells. Fatty acid synthesis provides building blocks of membrane growth, provides substrates for acid oxidation, the main energy source for CaP cells, provides building blocks for anti-apoptotic and proinflammatory molecules, and provides molecules that stimulate steroidogenesis. In this project it has been shown that insulin upregulates FASN and ACC, which synthesize fatty acids, as well as upregulating hormone sensitive lipase (HSL), diazepam-binding inhibitor (DBI), and long-chain acyl-CoA synthetase 3 (ACSL3), which contribute to lipid activation of steroidogenesis. Insulin also upregulates total lipid levels and de novo lipogenesis, which can be suppressed by inhibition of the insulin receptor (INSR). The fatty acids synthesized after insulin treatment are those that have been associated with CaP; furthermore, microarray data suggests insulin may upregulate fatty acid biosynthesis, metabolism and arachidonic acid metabolism pathways, which have been implicated in CaP growth and survival. Pharmacological agents used to treat patients with hyperinsulinemia/ hyperlipidemia have gained much interest in regards to CaP risk and treatment; however, the scientific rationale behind these clinical applications has not been examined. This thesis explores whether the use of metformin or simvastatin would decrease either lipogenesis or steroidogenesis or both in CaP cells. Simvastatin is a 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) inhibitor, which blocks synthesis of cholesterol, the building block of steroids/ androgens. It has also been postulated to down regulate SREBP in other metabolic disorders. It has been shown in this thesis, in LNCaP cells, that simvastatin inhibited and decreased insulin induced steroidogenesis and lipogenesis, respectively, but increased these pathways in the absence of insulin. Conversely, metformin, which activates AMP-activated protein kinase (AMPK) to shut down lipogenesis, cholesterol synthesis, and protein synthesis, highly suppresses both steroidogenesis and lipogenesis in the presence and absence of insulin. Lastly, because it has been demonstrated to increase steroidogenesis in other cell lines, and because the elucidation of any factors affecting steroidogenesis is important to understanding CaP, the effect of IGF2 on steroidogenesis in CaP cells was investigated. In patient samples, as men progress to CRPC, IGF2 mRNA and the protein levels of the receptors it may signal through are upregulated. It has also been demonstrated that IGF2 upregulates steroidogenic enzymes at both the mRNA and protein levels in LNCaP cells, increases intracellular and secreted steroid/androgen levels in LNCaPs to levels sufficient to stimulate the AR, and upregulated de novo steroidogenesis in LNCaPs and VCaPs. As well, inhibition of INSR and insulin-like growth factor 1 receptor (IGF1R), which IGF2 signals through, suggests that induction of steroidogenesis may be occurring predominantly through IGF1R. In summary, this project has illuminated for the first time that insulin is likely to play a large role in cancer progression, through upregulation of the steroidogenesis and lipogenesis pathways at the mRNA and protein levels, and production levels, and demonstrates a novel role for IGF-II in CaP progression through stimulation of steroidogenesis. It has also been demonstrated that metformin and simvastatin drugs may be useful in suppressing the insulin induction of these pathways. This project affirms the pathways by which ADT- induced metabolic syndrome may exacerbate CaP progression and strongly suggests that the monitoring and modulation of the metabolic state of CaP patients could have a strong impact on their therapeutic outcomes.

The FAM deubiquitylating enzyme localizes to multiple points of protein trafficking in epithelia, where it associates with E-cadherin and β-catenin

Ubiquitylation is a necessary step in the endocytosis and lysosomal trafficking of many plasma membrane proteins and can also influence protein trafficking in the biosynthetic pathway. Although a molecular understanding of ubiquitylation in these processes is beginning to emerge, very little is known about the role deubiquitylation may play. Fat Facets in mouse (FAM) is substrate-specific deubiquitylating enzyme highly expressed in epithelia where it interacts with its substrate, β-catenin. Here we show, in the polarized intestinal epithelial cell line T84, FAM localized to multiple points of protein trafficking. FAM interacted with β-catenin and E-cadherin in T84 cells but only in subconfluent cultures. FAM extensively colocalized with β-catenin in cytoplasmic puncta but not at sites of cell-cell contact as well as immunoprecipitating with β-catenin and E-cadherin from a higher molecular weight complex (~500 kDa). At confluence FAM neither colocalized with, nor immunoprecipitated, β-catenin or E-cadherin, which were predominantly in a larger molecular weight complex (~2 MDa) at the cell surface. Overexpression of FAM in MCF-7 epithelial cells resulted in increased β-catenin levels, which localized to the plasma membrane. Expression of E-cadherin in L-cell fibroblasts resulted in the relocalization of FAM from the Golgi to cytoplasmic puncta. These data strongly suggest that FAM associates with E-cadherin and β-catenin during trafficking to the plasma membrane.

Advanced glycation endproducts in horses with insulin-induced laminitis

Advanced glycation endproducts (AGEs) have been implicated in the pathogenesis of cancer, inflammatory conditions and diabetic complications. An interaction of AGEs with their receptor (RAGE) results in increased release of pro-inflammatory cytokines and reactive oxygen species (ROS), causing damage to susceptible tissues. Laminitis, a debilitating foot condition of horses, occurs in association with endocrine dysfunction and the potential involvement of AGE and RAGE in the pathogenesis of the disease has not been previously investigated. Glucose transport in lamellar tissue is thought to be largely insulin-independent (GLUT-1), which may make the lamellae susceptible to protein glycosylation and oxidative stress during periods of increased glucose metabolism. Archived lamellar tissue from horses with insulin-induced laminitis (n=4), normal control horses (n=4) and horses in the developmental stages (6 h, 12 h and 24 h) of the disease (n=12) was assessed for AGE accumulation and the presence of oxidative protein damage and cellular lipid peroxidation. The equine-specific RAGE gene was identified in lamellar tissue, sequenced and is now available on GenBank. Lamellar glucose transporter (GLUT-1 and GLUT-4) gene expression was assessed quantitatively with qRT-PCR in laminitic and control horses and horses in the mid-developmental time-point (24 h) of the disease. Significant AGE accumulation had occurred by the onset of insulin-induced laminitis (48 h) but not at earlier time-points, or in control horses. Evidence of oxidative stress was not found in any group. The equine-specific RAGE gene was not expressed differently in treated and control animals, nor was the insulin-dependent glucose transporter GLUT-4. However, the glucose transporter GLUT-1 was increased in lamellar tissue in the developmental stages of insulin-induced laminitis compared to control horses and the insulin-independent nature of the lamellae may facilitate AGE formation. However, due to the lack of AGE accumulation during disease development and a failure to detect an increase in ROS or upregulation of RAGE, it appears unlikely that oxidative stress and protein glycosylation play a central role in the pathogenesis of acute, insulin-induced laminitis.

Cellular settings mediating Src Substrate switching between Focal Adhesion Kinase Tyrosine 861 and CUB-domain-containing protein 1 (CDCP1) Tyrosine 734

Reciprocal interactions between Src family kinases (SFKs) and focal adhesion kinase (FAK) are critical during changes in cell attachment. Recently it has been recognized that another SFK substrate, CUB-domain-containing protein 1 (CDCP1), is differentially phosphorylated during these events. However, the molecular processes underlying SFK-mediated phosphorylation of CDCP1 are poorly understood. Here we identify a novel mechanism in which FAK tyrosine 861 and CDCP1-Tyr-734 compete as SFK substrates and demonstrate cellular settings in which SFKs switch between these sites. Our results show that stable CDCP1 expression induces robust SFK-mediated phosphorylation of CDCP1-Tyr-734 with concomitant loss of p-FAK-Tyr-861 in adherent HeLa cells. SFK substrate switching in these cells is dependent on the level of expression of CDCP1 and is also dependent on CDCP1-Tyr-734 but is independent of CDCP1-Tyr-743 and -Tyr-762. In HeLa CDCP1 cells, engagement of SFKs with CDCP1 is accompanied by an increase in phosphorylation of Src-Tyr-416 and a change in cell morphology to a fibroblastic appearance dependent on CDCP1-Tyr-734. SFK switching between FAK-Tyr-861 and CDCP1-Tyr-734 also occurs during changes in adhesion of colorectal cancer cell lines endogenously expressing these two proteins. Consistently, increased p-FAK-Tyr-861 levels and a more epithelial morphology are seen in colon cancer SW480 cells silenced for CDCP1. Unlike protein kinase Cδ, FAK does not appear to form a trimeric complex with Src and CDCP1. These data demonstrate novel aspects of the dynamics of SFK-mediated cell signaling that may be relevant during cancer progression.