122 resultados para ATP depletion

em Queensland University of Technology - ePrints Archive


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1. Both dietary magnesium depletion and potassium depletion (confirmed by tissue analysis) were induced in rats which were then compared with rats treated with chlorothiazide (250 mg/kg diet) and rats on a control synthetic diet. 2. Brain and muscle intracellular pH was measured by using a surface coil and [31P]-NMR to measure the chemical shift of inorganic phosphate. pH was also measured in isolated perfused hearts from control and magnesium-deficient rats. Intracellular magnesium status was assessed by measuring the chemical shift of β-ATP in brain. 3. There was no evidence for magnesium deficiency in the chlorothiazide-treated rats on tissue analysis or on chemical shift of β-ATP in brain. Both magnesium and potassium deficiency, but not chlorothiazide treatment, were associated with an extracellular alkalosis. 4. Magnesium deficiency led to an intracellular alkalosis in brain, muscle and heart. Chlorothiazide treatment led to an alkalosis in brain. Potassium deficiency was associated with a normal intracellular pH in brain and muscle. 5. Magnesium depletion and chlorothiazide treatment produce intracellular alkalosis by unknown mechanism(s).

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Prior in vitro studies, utilizing 31Pn uclear magnetic resonance (31PN MR) to measure the chemical shift (CT) of 0-ATP and lengthening of the phosphocreatine spin-spin (7"') relaxation time, suggested an assessment of their efficacy in measuring magnesium depletion in vivo. Dietary magnesium depletion (Me$) produced markedly lower magnesium in plasma (0.44 vs 1. I3 mmol/liter) and bone (1 30 vs 190 pmol/g) but much smaller changes in muscle (41 vs 45 pmol/g, P < 0.01), heart (42.5 vs 44.6 prnol/g), and brain (30 vs 32 pmollg). NMR experiments in anesthetized rats in a Bruker 7-T vertical bore magnet showed that in M e $ rats there was a significant change in brain j3-ATP shift (16.15 vs 16.03 ppm, P < 0.05). These chemical shifts gave a calculated free [Mg"] of 0.71 mM (control) and 0.48 mM (MgZ+$). In muscle the change in j3-ATP shift was not significant (Me$ 15.99 ppm, controls 15.96 ppm), corresponding to a calculated free M P of 0.83 and 0.95 mM, respectively. Phosphccreatine Tz (Carr-Purcell, spin-echo pulse sequence) was no different with M e $ in muscle in vivo (surface coil) (M$+$ 136, control 142 ms) or in isolated perfused hearts (Helmholtz coil) (control 83, M e $ 92 ms). 3'P NMR is severely limited in its ability to detect dietary magnesium depletion in vivo. Measurement of j3-ATP shift in brain may allow studies of the effects of interaction in group studies but does not allow prediction of an individual magnesium status.

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This paper presents a database ATP (Alternative Transient Program) simulated waveforms for shunt reactor switching cases with vacuum breakers in motor circuits following interruption of the starting current. The targeted objective is to provide multiple reignition simulated data for diagnostic and prognostic algorithms development, but also to help ATP users with practical study cases and component data compilation for shunt reactor switching. This method can be easily applied with different data for the different dielectric curves of circuit-breakers and networks. This paper presents design details, discusses some of the available cases and the advantages of such simulated data.

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Background: It has been proposed that adenosine triphosphate (ATP) released from red blood cells (RBCs) may contribute to the tight coupling between blood flow and oxygen demand in contracting skeletal muscle. To determine whether ATP may contribute to the vasodilatory response to exercise in the forearm, we measured arterialised and venous plasma ATP concentration and venous oxygen content in 10 healthy young males at rest, and at 30 and 180 seconds during dynamic handgrip exercise at 45% of maximum voluntary contraction (MVC). Results: Venous plasma ATP concentration was elevated above rest after 30 seconds of exercise (P < 0.05), and remained at this higher level 180 seconds into exercise (P < 0.05 versus rest). The increase in ATP was mirrored by a decrease in venous oxygen content. While there was no significant relationship between ATP concentration and venous oxygen content at 30 seconds of exercise, they were moderately and inversely correlated at 180 seconds of exercise (r = -0.651, P = 0.021). Arterial ATP concentration remained unchanged throughout exercise, resulting in an increase in the venous-arterial ATP difference. Conclusions: Collectively these results indicate that ATP in the plasma originated from the muscle microcirculation, and are consistent with the notion that deoxygenation of the blood perfusing the muscle acts as a stimulus for ATP release. That ATP concentration was elevated just 30 seconds after the onset of exercise also suggests that ATP may be a contributing factor to the blood flow response in the transition from rest to steady state exercise.

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Circuit breaker restrikes are unwanted occurrence, which can ultimately lead to breaker. Before 2008, there was little evidence in the literature of monitoring techniques based on restrike measurement and interpretation produced during switching of capacitor banks and shunt reactor banks. In 2008 a non-intrusive radiometric restrike measurement method, as well a restrike hardware detection algorithm was developed. The limitations of the radiometric measurement method are a band limited frequency response as well as limitations in amplitude determination. Current detection methods and algorithms required the use of wide bandwidth current transformers and voltage dividers. A novel non-intrusive restrike diagnostic algorithm using ATP (Alternative Transient Program) and wavelet transforms is proposed. Wavelet transforms are the most common use in signal processing, which is divided into two tests, i.e. restrike detection and energy level based on deteriorated waveforms in different types of restrike. A ‘db5’ wavelet was selected in the tests as it gave a 97% correct diagnostic rate evaluated using a database of diagnostic signatures. This was also tested using restrike waveforms simulated under different network parameters which gave a 92% correct diagnostic responses. The diagnostic technique and methodology developed in this research can be applied to any power monitoring system with slight modification for restrike detection.

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BACKGROUND The transgenic adenocarcinoma of the mouse prostate (TRAMP) model closely mimics PC-progression as it occurs in humans. However, the timing of disease incidence and progression (especially late stage) makes it logistically difficult to conduct experiments synchronously and economically. The development and characterization of androgen depletion independent (ADI) TRAMP sublines are reported. METHODS Sublines were derived from androgen-sensitive TRAMP-C1 and TRAMP-C2 cell lines by androgen deprivation in vitro and in vivo. Epithelial origin (cytokeratin) and expression of late stage biomarkers (E-cadherin and KAI-1) were evaluated using immunohistochemistry. Androgen receptor (AR) status was assessed through quantitative real time PCR, Western blotting, and immunohistochemistry. Coexpression of AR and E-cadherin was also evaluated. Clonogenicity and invasive potential were measured by soft agar and matrigel invasion assays. Proliferation/survival of sublines in response to androgen was assessed by WST-1 assay. In vivo growth of subcutaneous tumors was assessed in castrated and sham-castrated C57BL/6 mice. RESULTS The sublines were epithelial and displayed ADI in vitro and in vivo. Compared to the parental lines, these showed (1) significantly faster growth rates in vitro and in vivo independent of androgen depletion, (2) greater tumorigenic, and invasive potential in vitro. All showed substantial downregulation in expression levels of tumor suppressor, E-cadherin, and metastatis suppressor, KAI-1. Interestingly, the percentage of cells expressing AR with downregulated E-cadherin was higher in ADI cells, suggesting a possible interaction between the two pathways. CONCLUSIONS The TRAMP model now encompasses ADI sublines potentially representing different phenotypes with increased tumorigenicity and invasiveness.

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We sought to identify fibroblast growth factor receptor 2 (FGFR2) kinase domain mutations that confer resistance to the pan-FGFR inhibitor, dovitinib, and explore the mechanism of action of the drug-resistant mutations. We cultured BaF3 cells overexpressing FGFR2 in high concentrations of dovitinib and identified fourteen dovitinib-resistant mutations, including the N550K mutation observed in 25% of FGFR2mutant endometrial cancers (EC). Structural and biochemical in vitro kinase analyses, together with BaF3 proliferation assays, showed that the resistance mutations elevate the intrinsic kinase activity of FGFR2. BaF3 lines were used to assess the ability of each mutation to confer cross-resistance to PD173074 and ponatinib. Unlike PD173074, ponatinib effectively inhibited all the dovitinib-resistant FGFR2 mutants except the V565I gatekeeper mutation, suggesting ponatinib but not dovitinib targets the active conformation of FGFR2 kinase. EC cell lines expressing wild-type FGFR2 were relatively resistant to all inhibitors. Whereas EC cell lines expressing mutated FGFR2 showed differential sensitivity. Within the FGFR2mutant cell lines, 3/7 showed marked resistance to PD173074 and relative resistance to dovitinib and ponatinib. This suggests that alternative mechanisms distinct from kinase domain mutations are responsible for intrinsic resistance in these three EC lines. Finally, overexpression of FGFR2N550K in JHUEM-2 cells (FGFR2C383R) conferred resistance (~5 fold) to PD173074, providing independent data that FGFR2N550K can be associated with drug resistance. Biochemical in vitro kinase analyses also shows ponatinib is more effective than dovitinib at inhibiting FGFR2N550K. We propose tumors harboring mutationally activated FGFRs should be treated with FGFR inhibitors that specifically bind the active kinase.

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We investigated the relationship between mitochondrial biogenesis, cell signalling and antioxidant enzymes by depleting skeletal muscle glutathione with diethyl maleate (DEM) which resulted in a demonstrable increase in oxidative stress during exercise. Animals were divided into six groups: (1) sedentary control rats; (2) sedentary rats treated with DEM; (3) exercise control rats euthanized immediately after exercise; (4) exercise rats + DEM; (5) exercise control rats euthanized 4 h after exercise, and; (6) exercise rats + DEM euthanized 4 h after exercise. Exercising animals ran on the treadmill at a 10% gradient at 20 m/min for the first 30 min. The speed was then increased every 10 min by 1.6 m/min until exhaustion. There was a reduction in total glutathione in the skeletal muscle of DEM treated animals compared to the control animals (P<0.05). Within the control group, total glutathione was higher in the sedentary group compared to after exercise (P<0.05). DEM treatment also significantly increased oxidative stress, as measured by increased plasma F2-isoprostanes (P<0.05). Exercising animals given DEM showed a significantly greater increase in peroxisome proliferator activated receptor γ coactivator-1α(PGC-1α) mRNA compared to the control animals that were exercised (P<0.05). This study provides novel evidence that by reducing the endogenous antioxidant glutathione in skeletal muscle and inducing oxidative stress through exercise, PGC-1α gene expression was augmented. These findings further highlight the important role of exercise induced oxidative stress in the regulation of mitochondrial biogenesis.

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Low density suburban development and excessive use of automobiles are associated with serious urban and environmental problems. These problems include traffic congestion, longer commuting times, high automobile dependency, air and water pollution, and increased depletion of natural resources. Master planned development suggests itself as a possible palliative for the ills of low density and high travel. The following study examines the patterns and dynamics of movement in a selection of master planned estates in Australia. The study develops new approaches for assessing the containment of travel within planned development. Its key aim is to clarify and map the relationships between trip generation and urban form and structure. The initial conceptual framework of the paper is developed in a review of literature related to urban form and travel behaviour. These concepts are tested empirically in a pilot study of suburban travel activity in master planned estates. A geographical information systems methodology is used to determine regional journey-to-work patterns and travel containment rates. Factors that influence selfcontainment patterns are estimated with a regression model. This research is a useful preliminary examination of travel self-containment in Australian master planned estates.

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Low density suburban development and excessive use of automobiles are associated with serious urban and environmental problems. These problems include traffic congestion, longer commuting times, high automobile dependency, air and water pollution, and increased depletion of natural resources. Master planned development suggests itself as a possible palliative for the ills of low density and high travel. The following study examines the patterns and dynamics of movement in a selection of master planned estates in Australia. The study develops new approaches for assessing the containment of travel within planned development. Its key aim is to clarify and map the relationships between trip generation and urban form and structure. The initial conceptual framework of the report is developed in a review of literature related to urban form and travel behaviour. These concepts are tested empirically in a pilot study of suburban travel activity in master planned estates. A geographical information systems (GIS) methodology is used to determine regional journey-to-work patterns and travel containment rates. Factors that influence self-containment patterns are estimated with a regression model. The key research findings of the pilot study are: - There is a strong relation between urban structural form and patterns of trip generation; - The travel self-containment of Australian master planned estates is lower than the scholarly literature implies would occur if appropriate planning principles to achieve sustainable urban travel were followed; - Proximity to the central business district, income level and education status are positively correlated with travel containment; - Master planned estates depend more on local and regional centres for employment than on the central business district; - The service sector is the major employer in and around master planned estates. It tends to provide part-time and casual employment rather than full-time employment; - Travel self-containment is negative correlated with car dependency. Master planned estates with less car dependent residents, and with good access to public transport, appear to be more self-contained and, consequently, more sustainable than the norm. This research is a useful preliminary examination of travel self-containment in Australian master planned estates. It by no means exhausts the subject. In future research we hope to further assess sustainable travel patterns with more detailed spatial analysis.

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- This paper presents a validation proposal for development of diagnostic and prognostic algorithms for SF6 puffer circuit-breakers reproduced from actual site waveforms. The re-ignition/restriking rates are duplicated in given circuits and the cumulative energy dissipated in interrupters by the restriking currents. The targeted objective is to provide a simulated database for diagnosis of re-ignition/restrikes relating to the phase to earth voltage and the number of re-ignition/restrikes as well as estimating the remaining life of SF6 circuit-breakers. The model-based diagnosis of a tool will be useful in monitoring re-ignition/restrikes as well as predicting a nozzle’s lifetime. This will help ATP users with practical study cases and component data compilation for shunt reactor switching and capacitor switching. This method can be easily applied with different data for the different dielectric curves of circuit breakers and networks. This paper presents modelling details and some of the available cases, required project support, the validation proposal, the specific plan for implementation and the propsed main contributions.

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It is known that adenosine 5'-triphosphate (ATP) is a cotransmitter in the heart. Additionally, ATP is released from ischemic and hypoxic myocytes. Therefore, cardiac-derived sources of ATP have the potential to modify cardiac function. ATP activates P2X(1-7) and P2Y(1-14) receptors; however, the presence of P2X and P2Y receptor subtypes in strategic cardiac locations such as the sinoatrial node has not been determined. An understanding of P2X and P2Y receptor localization would facilitate investigation of purine receptor function in the heart. Therefore, we used quantitative PCR and in situ hybridization to measure the expression of mRNA of all known purine receptors in rat left ventricle, right atrium and sinoatrial node (SAN), and human right atrium and SAN. Expression of mRNA for all the cloned P2 receptors was observed in the ventricles, atria, and SAN of the rat. However, their abundance varied in different regions of the heart. P2X(5) was the most abundant of the P2X receptors in all three regions of the rat heart. In rat left ventricle, P2Y(1), P2Y(2), and P2Y(14) mRNA levels were highest for P2Y receptors, while in right atrium and SAN, P2Y(2) and P2Y(14) levels were highest, respectively. We extended these studies to investigate P2X(4) receptor mRNA in heart from rats with coronary artery ligation-induced heart failure. P2X(4) receptor mRNA was upregulated by 93% in SAN (P < 0.05), while a trend towards an increase was also observed in the right atrium and left ventricle (not significant). Thus, P2X(4)-mediated effects might be modulated in heart failure. mRNA for P2X(4-7) and P2Y(1,2,4,6,12-14), but not P2X(2,3) and P2Y(11), was detected in human right atrium and SAN. In addition, mRNA for P2X(1) was detected in human SAN but not human right atrium. In human right atrium and SAN, P2X(4) and P2X(7) mRNA was the highest for P2X receptors. P2Y(1) and P2Y(2) mRNA were the most abundant for P2Y receptors in the right atrium, while P2Y(1), P2Y(2), and P2Y(14) were the most abundant P2Y receptor subtypes in human SAN. This study shows a widespread distribution of P2 receptor mRNA in rat heart tissues but a more restricted presence and distribution of P2 receptor mRNA in human atrium and SAN. This study provides further direction for the elucidation of P2 receptor modulation of heart rate and contractility.

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In recent years, cities show increasing signs of environmental problems due to the negative impacts of urban activities. The degradation and depletion of natural resources, climate change and development pressure on green areas have become major concerns for cities. In response to these problems, urban planning policies have shifted to a sustainable focus and authorities have begun to develop new strategies for improving the quality of urban ecosystems. An extremely important function of an urban ecosystem is to provide healthy and sustainable environments for both natural systems and communities. Therefore, ecological planning is a functional requirement in the establishment of sustainable built environment. With ecological planning human needs are supplied while natural resources are used in the most effective and sustainable manner. And the maintenance of ecological balance is sustained. Protecting human and environmental health, having healthy ecosystems, reducing environmental pollution and providing green spaces are just a few of the many benefits of ecological planning. In this context, the paper briefly presents a short overview of the importance of the implementation of ecological planning into sustainable urban development. Furthermore, the paper defines the conceptual framework of a new method for developing sustainable urban ecosystems through ecological planning approach. In the future of the research, this model will be developed as a guideline for the assessment of the ecological sustainability in built environments.

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Two areas of particular importance in prostate cancer progression are primary tumour development and metastasis. These processes involve a number of physiological events, the mediators of which are still being discovered and characterised. Serine proteases have been shown to play a major role in cancer invasion and metastasis. The recently discovered phenomenon of their activation of a receptor family known as the protease activated receptors (PARs) has extended their physiological role to that of signaling molecule. Several serine proteases are expressed by malignant prostate cancer cells, including members of the kallikreinrelated peptidase (KLK) serine protease family, and increasingly these are being shown to be associated with prostate cancer progression. KLK4 is highly expressed in the prostate and expression levels increase during prostate cancer progression. Critically, recent studies have implicated KLK4 in processes associated with cancer. For example, the ectopic over-expression of KLK4 in prostate cancer cell lines results in an increased ability of these cells to form colonies, proliferate and migrate. In addition, it has been demonstrated that KLK4 is a potential mediator of cellular interactions between prostate cancer cells and osteoblasts (bone forming cells). The ability of KLK4 to influence cellular behaviour is believed to be through the selective cleavage of specific substrates. Identification of relevant in vivo substrates of KLK4 is critical to understanding the pathophysiological roles of this enzyme. Significantly, recent reports have demonstrated that several members of the KLK family are able to activate PARs. The PARs are relatively new members of the seven transmembrane domain containing G protein coupled receptor (GPCR) family. PARs are activated through proteolytic cleavage of their N-terminus by serine proteases, the resulting nascent N-terminal binds intramolecularly to initiate receptor activation. PARs are involved in a number of patho-physiological processes, including vascular repair and inflammation, and a growing body of evidence suggests roles in cancer. While expression of PAR family members has been documented in several types of cancers, including prostate, the role of these GPCRs in prostate cancer development and progression is yet to be examined. Interestingly, several studies have suggested potential roles in cellular invasion through the induction of cytoskeletal reorganisation and expression of basement membrane-degrading enzymes. Accordingly, this program of research focussed on the activation of the PARs by the prostate cancer associated enzyme KLK4, cellular processing of activated PARs and the expression pattern of receptor and agonist in prostate cancer. For these studies KLK4 was purified from the conditioned media of stably transfected Sf9 insect cells expressing a construct containing the complete human KLK4 coding sequence in frame with a V5 epitope and poly-histidine encoding sequences. The first aspect of this study was the further characterisation of this recombinant zymogen form of KLK4. The recombinant KLK4 zymogen was demonstrated to be activatable by the metalloendopeptidase thermolysin and amino terminal sequencing indicated that thermolysin activated KLK4 had the predicted N-terminus of mature active KLK4 (31IINED). Critically, removal of the pro-region successfully generated a catalytically active enzyme, with comparable activity to a previously published recombinant KLK4 produced from S2 insect cells. The second aspect of this study was the activation of the PARs by KLK4 and the initiation of signal transduction. This study demonstrated that KLK4 can activate PAR-1 and PAR-2 to mobilise intracellular Ca2+, but failed to activate PAR-4. Further, KLK4 activated PAR-1 and PAR-2 over distinct concentration ranges, with KLK4 activation and mobilisation of Ca2+ demonstrating higher efficacy through PAR-2. Thus, the remainder of this study focussed on PAR-2. KLK4 was demonstrated to directly cleave a synthetic peptide that mimicked the PAR-2 Nterminal activation sequence. Further, KLK4 mediated Ca2+ mobilisation through PAR-2 was accompanied by the initiation of the extra-cellular regulated kinase (ERK) cascade. The specificity of intracellular signaling mediated through PAR-2 by KLK4 activation was demonstrated by siRNA mediated protein depletion, with a reduction in PAR-2 protein levels correlating to a reduction in KLK4 mediated Ca2+mobilisation and ERK phosphorylation. The third aspect of this study examined cellular processing of KLK4 activated PAR- 2 in a prostate cancer cell line. PAR-2 was demonstrated to be expressed by five prostate derived cell lines including the prostate cancer cell line PC-3. It was also demonstrated by flow cytometry and confocal microscopy analyses that activation of PC-3 cell surface PAR-2 by KLK4 leads to internalisation of this receptor in a time dependent manner. Critically, in vivo relevance of the interaction between KLK4 and PAR-2 was established by the observation of the co-expression of receptor and agonist in primary prostate cancer and prostate cancer bone lesion samples by immunohistochemical analysis. Based on the results of this study a number of exciting future studies have been proposed, including, delineating differences in KLK4 cellular signaling via PAR-1 and PAR-2 and the role of PAR-1 and PAR-2 activation by KLK4 in prostate cancer cells and bone cells in prostate cancer progression.