Phosphatidylinositol 3-kinase C2 alpha is essential for ATP-dependent priming of neurosecretory granule exocytosis

Neurotransmitter release and hormonal secretion are highly regulated processes culminating in the calcium-dependent fusion of secretory vesicles with the plasma membrane. Here, we have identified a role for phosphatidylinositol 3-kinase C2 alpha (PI3K-C2 alpha) and its main catalytic product, PtdIns3P, in regulated exocytosis. In neuroendocrine cells, PI3K-C2 alpha is present on a subpopulation of mature secretory granules. Impairment of PI3K-C2 alpha function specifically inhibits the ATP-dependent priming phase of exocytosis. Overexpression of wild-type PI3K-C2 alpha enhanced secretion, whereas transfection of PC12 cells with a catalytically inactive PI3K-C2 alpha mutant or a 2xFYVE domain sequestering PtdIns3P abolished secretion. Based on these results, we propose that production of PtdIns3P by PI3K-C2 alpha is required for acquisition of fusion competence in neurosecretion.

Activation of ATP-ubiquitin-dependent proteolysis in skeletal muscle in vivo and murine myoblasts in vitro by a proteolysis-inducing factor (PIF)

Loss of skeletal muscle is a major factor in the poor survival of patients with cancer cachexia. This study examines the mechanism of catabolism of skeletal muscle by a tumour product, proteolysis-inducing factor (PIF). Intravenous administration of PIF to normal mice produced a rapid decrease in body weight (1.55 ± 0.12 g in 24 h) that was accompanied by increased mRNA levels for ubiquitin, the Mr 14 000 ubiquitin carrier-protein, E2, and the C9 proteasome subunit in gastrocnemius muscle. There was also increased protein levels of the 20S proteasome core and 19S regulatory subunit, detectable by immunoblotting, suggesting activation of the ATP-ubiquitin-dependent proteolytic pathway. An increased protein catabolism was also seen in C2C12 myoblasts within 24 h of PIF addition with a bell-shaped dose-response curve and a maximal effect at 2-4 nM. The enhanced protein degradation was attenuated by anti-PIF antibody and by the proteasome inhibitors MG115 and lactacystin. Glycerol gradient analysis of proteasomes from PIF-treated cells showed an elevation in chymotrypsin-like activity, while Western analysis showed a dose-related increase in expression of MSSI, an ATPase that is a regulatory subunit of the proteasome, with a dose-response curve similar to that for protein degradation. These results confirm that PIF acts directly to stimulate the proteasome pathway in muscle cells and may play a pivotal role in protein catabolism in cancer cachexia. © 2001 Cancer Research Campaign.

The measurement and significance of ATP pools in activated sludge treating coke oven liquors

Coke oven liquor is a toxic wastewater produced in large quantities by the Iron and Steel, and Coking Industries, and gives rise to major effluent treatment problems in those industries. Conscious of the potentially serious environmental impact of the discharge of such wastes, pollution control agencies in many countries have made progressively more stringent quality requirements for the discharge of the treated waste. The most common means of treating the waste is the activated sludge process. Problems with achieving consistently satisfactory treatment by this process have been experienced in the past. The need to improve the quality of the discharge of the treated waste prompted attempts by TOMLINS to model the process using Adenosine Triphosophnte (ATP) as a measure of biomass, but these were unsuccessful. This thesis describes work that was carried out to determine the significance of ATP in the activated sludge treatment of the waste. The use of ATP measurements in wastewater treatment were reviewed. Investigations were conducted into the ATP behaviour of the batch activated sludge treatment of two major components of the waste, phenol, and thiocyanate, and the continuous activated sludge treatment of the liquor itself, using laboratory scale apparatus. On the basis of these results equations were formulated to describe the significance of ATP as a measured activity and biomass in the treatment system. These were used as the basis for proposals to use ATP as a control parameter in the activated sludge treatment of coke oven liquor, and wastewaters in general. These had relevance both to the treatment of the waste in the reactor and to the settlement of the sludge produced in the secondary settlement stage of the treatment process.

Impact of ATP-binding cassette, subfamily B, member 1 pharmacogenetics on tacrolimus-associated nephrotoxicity and dosage requirements in paediatric patients with liver transplant

Importance of the field: Tacrolimus is the most commonly used immunosuppressive agent following solid-organ transplantation in children. Its clinical use, however, is complicated by side effects (mainly nephrotoxicity), narrow therapeutic index and pharmacokinetic variability which can result in an increased risk of treatment failure or toxicity. Studies examining interindividual differences in the expression of the ABCB1 (ATP-binding cassette, subfamily B, member 1) gene (which encodes the drug transporter, P-gp) and its genetic polymorphisms have attempted to elucidate variations in tacrolimus response and disposition in children. Areas covered in this review: This review explores pharmacogenetic knowledge developed over the last decade regarding the impact of ABCB1 polymorphisms on tacrolimus toxicity and dosage requirements in children. What the reader will gain: A better understanding of the role of ABCB1 genetic polymorphisms (and corresponding haplotypes) and ABCB1 expression levels in various tissues and organs on tacrolimus outcomes in children with liver transplant. Take home message: Pharmacogenetics offers significant potential for optimising tacrolimus use. ABCB1 donor genotypes and ABCB1 expression level in the intestine and leukocytes may be useful in dosage selection. Large prospective studies are, however, required to further explore the potential of genetic testing in identifying children who are at risk of toxicity and to better individualise tacrolimus therapy.

A comparison of the effects of ocular preservatives on mammalian and microbial ATP and glutathione levels

The aim of this study was to investigate the mechanism of action of the preservative sodium chlorite (NaClO2), and the relationship with intracellular glutathione depletion. A detailed comparison of the dose responses of two cultured ocular epithelial cell types and four species of microorganism was carried out, and comparisons were also made with the quaternary ammonium compound benzalkonium chloride (BAK), and the oxidant hydrogen peroxide (H2O2). The viability of mammalian and microbial cells was assessed in the same way, by the measurement of intracellular ATP using a bioluminescence method. Intracellular total glutathione was measured by reaction with 5,5'-dithiobis-2-nitrobenzoic acid in a glutathione reductase-dependent recycling assay. BAK and H2O2 caused complete toxicity to conjunctival and corneal epithelial cells at similar to25 ppm, in contrast to NaClO2 , where >100 ppm was required. The fungi Candida albicans and Alternaria alternata had a higher resistance to NaClO2 than the bacteria Staphyloccus aureus and Pseudomonas aeruginosa , but the bacteria were extremely resistant to H2O2 NaClO2 caused substantial depletion of intracellular glutathione in all cell types, at concentrations ranging from <10 ppm in Pseudomonas , 25-100 ppm in epithelial cells, to >500 ppm in fungal cells. The mechanisms of cytotoxicity of NaClO2 , H2O2 and BAK all appeared to differ. NaClO2 was found to have the best balance of high antibacterial toxicity with low ocular toxicity. The lower toxicity of NaClO2 to the ocular cells, compared with BAK and H2O2 , is in agreement with fewer reported adverse effects of application in the eye.

Detergent-free purification of ABC (ATP-binding-cassette) transporters

ABC (ATP-binding-cassette) transporters carry out many vital functions and are involved in numerous diseases, but study of the structure and function of these proteins is often hampered by their large size and membrane location. Membrane protein purification usually utilizes detergents to solubilize the protein from the membrane, effectively removing it from its native lipid environment. Subsequently, lipids have to be added back and detergent removed to reconstitute the protein into a lipid bilayer. In the present study, we present the application of a new methodology for the extraction and purification of ABC transporters without the use of detergent, instead, using a copolymer, SMA (polystyrene-co-maleic acid). SMA inserts into a bilayer and assembles into discrete particles, essentially solubilizing the membrane into small discs of bilayer encircled by a polymer, termed SMALPs (SMA lipid particles). We show that this polymer can extract several eukaryotic ABC transporters, P-glycoprotein (ABCB1), MRP1 (multidrug-resistance protein 1; ABCC1), MRP4 (ABCC4), ABCG2 and CFTR (cystic fibrosis transmembrane conductance regulator; ABCC7), from a range of different expression systems. The SMALP-encapsulated ABC transporters can be purified by affinity chromatography, and are able to bind ligands comparably with those in native membranes or detergent micelles. A greater degree of purity and enhanced stability is seen compared with detergent solubilization. The present study demonstrates that eukaryotic ABC transporters can be extracted and purified without ever being removed from their lipid bilayer environment, opening up awide range of possibilities for the future study of their structure and function. © The Authors Journal compilation © 2014 Biochemical Society.

Phospholipid chlorohydrins cause ATP depletion and toxicity in human myeloid cells

Chlorohydrins of stearoyl-oleoyl phosphatidylcholine (SOPC), stearoyl-linoleoyl phosphatidylcholine, and stearoyl-arachidonyl phosphatidylcholine were incubated with cultured myeloid cells (111,60) for 24 h, and the cellular ATP level was measured using a bioluminescent assay. The chlorohydrins caused significant depletion of cellular ATP in the range 10100 muM. The ATP depletion by the phospholipid chlorohydrins was slightly less than that of 4-hydroxy-2-nonenal, but greater than that of hexanal, trans-2-nonenal, and autoxidised palmitoyl-arachidonoyl phosphatidylcholine. SOPC chlorohydrin was also found to cause loss of viability in U937 cells, and thus phospholipid chlorohydrins could contribute to the formation of a necrotic core in advanced atherosclerotic lesions.

Modelagem e análise dinâmica de um sistema de conversão de energia eólica dotado de gerador síncrono de imã permanente utilizando a plataforma ATP

Human development requires a broad balance between ecological, social and economic factors in order to ensure its own sustainability. In this sense, the search for new sources of energy generation, with low deployment and operation costs, which cause the least possible impact to the environment, has been the focus of attention of all society segments. To do so, the reduction in exploration of fossil fuels and the encouragement of using renewable energy resources for distributed generation have proved interesting alternatives to the expansion of the energy matrix of various countries in the world. In this sense, the wind energy has acquired an increasingly significant role, presenting increasing rates of power grid penetration and highlighting technological innovations such as the use of permanent magnet synchronous generators (PMSG). In Brazil, this fact has also been noted and, as a result, the impact of the inclusion of this source in the distribution and sub-transmission power grid has been a major concern of utilities and agents connected to Brazilian electrical sector. Thus, it is relevant the development of appropriate computational tools that allow detailed predictive studies about the dynamic behavior of wind farms, either operating with isolated load, either connected to the main grid, taking also into account the implementation of control strategies for active/reactive power generation and the keeping of adequate levels of voltage and frequency. This work fits in this context since it comprises mathematical and computational developments of a complete wind energy conversion system (WECS) endowed with PMSG using time domain techniques of Alternative Transients Program (ATP), which prides itself a recognized reputation by scientific and academic communities as well as by electricity professionals in Brazil and elsewhere. The modeling procedures performed allowed the elaboration of blocks representing each of the elements of a real WECS, comprising the primary source (the wind), the wind turbine, the PMSG, the frequency converter, the step up transformer, the load composition and the power grid equivalent. Special attention is also given to the implementation of wind turbine control techniques, mainly the pitch control responsible for keeping the generator under the maximum power operation point, and the vector theory that aims at adjusting the active/reactive power flow between the wind turbine and the power grid. Several simulations are performed to investigate the dynamic behavior of the wind farm when subjected to different operating conditions and/or on the occurrence of wind intensity variations. The results have shown the effectiveness of both mathematical and computational modeling developed for the wind turbine and the associated controls.

Régulation moléculaire et cellulaire de l'efflux de cholestérol par le transporteur ATP-binding cassette A1(ABCA1)

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Contribution à l'étude du mécanisme de sécrétion d'ATP par des cellules épithéliales pulmonaires et des fibroblastes soumis à un choc hypotonique

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Régulation moléculaire et cellulaire de l'efflux de cholestérol par le transporteur ATP-binding cassette A1(ABCA1)

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Contribution à l'étude du mécanisme de sécrétion d'ATP par des cellules épithéliales pulmonaires et des fibroblastes soumis à un choc hypotonique

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

ATP content in surface sediments of the Northeast Atlantic (Table 1)

1. ATP in deep-sea sediments can be determined after it is adsorbed on a mixture of the sediment and calcium carbonate by measuring the luminescence of the reaction of the mixture and luciferin-luciferase. 2. ATP contents of the toplayer of northeastern Atlantic sediments (Josephine Bank and northern Canary Basin) decrease with increasing depths of 252, 408, 1445, 1769, 2149, 4897, 5510m: 0.96, 0.61, 0.13, 0.10, 0.21, 0.05, 0.07 µg ATP/ml wet sediment. The decreasing values are in accordance with the decrease of macrobenthos and meiobenthos biomass in the deep-sea. 3. The ATP content of deep-sea nematodes is about 1 ? of their wet weight. 4. At the two deepest stations, less than 50% of the ATP measured in the sediment is represented by nematodes, copepods, other "hard" meiofauna groups and bacteria.