Membrane-bound alkaline phosphatase from ectopic mineralization and rat bone marrow cell culture

Cells from rat bone marrow exhibit the proliferation-differentiation sequence of osteoblasts, form mineralized extracellular matrix in vitro and release alkaline phosphatase into the medium. Membrane-bound alkaline phosphatase was obtained by method that is easy to reproduce, simpler and fast when compared with the method used to obtain the enzyme from rat osseous plate. The membrane-bound alkaline phosphatase from cultures of rat bone marrow cells has a MWr of about 120 kDa and specific PNPP activity of 1200 U/tng. The ecto-enzyme is anchored to the plasma membrane by the GPI anchor and can be released by PIPLC (selective treatment) or polidocanol (0.2 mg/mL protein and 1% (w/v) detergent). The apparent optimum pH for PNPP hydrolysis by the enzyme was pH 10. This fraction hydrolyzes ATP (240 U/mg), ADP (350 U/ mg), glucose 1-phosphate (1100 U/mg), glucose 6-phosphate (340 Wing), fructose 6-phosphate (460 U/mg), pyrophosphate (330 U/mg) and (3glycerophosphate (600 U/mg). Cooperative effects were observed for the hydrolysis of PPi and beta-glycerophosphate. PNPPase activity was inhibited by 0.1 mM vanadate (46%), 0.1 mM ZnCl2 (68%), 1 mM levamisole (66%), 1 mM arsenate (44%), 10 mM phosphate (21%) and 1 mM theophylline (72%). We report the biochemical characterization of membrane-bound alkaline phosphatase obtained from rat bone marrow cells cultures, using a method that is simple, rapid and easy to reproduce. Its properties are compared with those of rat osseous plate enzyme and revealed that the alkaline phosphatase obtained has some kinetics and structural behaviors with higher levels of enzymatic activity, facilitating the comprehension of the mineralization process and its function. (c) 2006 Elsevier B.V. All rights reserved.

Construction of an alkaline phosphatase-liposome system: a tool for biomineralization study

Alkaline phosphatase is required for the mineralization of bone and cartilage. This enzyme is localized in the matrix vesicle, which plays a role key in calcifying cartilage. In this paper. we standardize a method for construction an alkaline phosphatase liposome system to mimic matrix vesicles and examine a some kinetic behavior of the incorporated enzyme. Polidocanol-solubilized alkaline phosphatase, free of detergent, was incorporated into liposomes constituted from dimyristoylphosphatidylcholine (DMPC), dilaurilphosphatidylcholine (DLPC) or dipalmitoylphosphatidylcholine (DPPC). This process was time-dependent and >95% of the enzyme was incorporated into the liposome after 4 h of incubation at 25 degreesC. Although, incorporation was more rapid when vesicles constituted from DPPC were used, the incorporation was more efficient using vesicles constituted from DMPC. The 395 nm diameter of the alkaline phosphatase-liposome system was relatively homogeneous and more stable when stored at 4 degreesC.Alkaline phosphatase was completely released from liposome system only using purified phosphatidylinositol-specific phospholipase C (PIPLC). These experiments confirm that the interaction between alkaline phosphatase and lipid bilayer of liposome is via GPI anchor of the enzyme, alone. An important point shown is that an enzyme bound to liposome does not lose the ability to hydrolyze ATP, pyrophosphate and p-nitrophenyl phosphate (PNPP), but a liposome environment affects its kinetic properties, specifically for pyrophosphate.The standardization of such system allows the study of the effect of phospholipids and the enzyme in in vitro and in vivo mineralization, since it reproduces many essential features of the matrix vesicle. (C) 2002 Elsevier B.V. Ltd. All rights reserved.

Erythrocyte ghost cell-alkaline phosphatase: construction and characterization of a vesicular system for use in biomineralization studies

Alkaline phosphatase is required for the mineralization of bone and cartilage. This enzyme is localized in the matrix vesicle, which plays a role key in calcifying cartilage. In this paper we standardize a method to construction a resealed ghost cell-alkaline phosphatase system to mimic matrix vesicles and examine the kinetic behavior of the incorporated enzyme. Polidocanol-solubilized alkaline phosphatase, free of detergent, was incorporated into resealed ghost cells. This process was time-dependent and practically 50% of the enzyme was incorporated into the vesicles in 40 h of incubation, at 25 degreesC. Alkaline phosphatase-ghost cell systems were relatively homogeneous with diameters of about 300 nm and were more stable when stored at -20 degreesC.Alkaline phosphatase was completely released from the resealed ghost cell-system using only phospholipase C. These experiments confirm that the interaction between alkaline phosphatase and the lipid bilayer of resealed ghost cell is exclusively via glycosylphosphatidylinositol (GPI) anchor of the enzyme.An important point shown is that an enzyme bound to resealed ghost cell does not lose the ability to hydrolyze ATP, pyrophosphate and p-nitrophenyl phosphate (PNPP), but the presence of a ghost membrane, as a support of the enzyme, affects its kinetic properties. Moreover, calcium ions stimulate and phosphate ions inhibit the PNPPase activity of alkaline phosphatase present in resealed ghost cells. (C) 2002 Elsevier B.V. B.V. All rights reserved.

Kinetic characterization of a membrane-specific ATPase from rat osseous plate and its possible significance on endochondral ossification

Treatment with phosphatidylinositol-specific phospholipase C of rat osseous plate membranes released up to 90-95% of alkaline phosphatase, but a specific ATPase activity (optimum pH = 7.5) remained bound to the membrane. The hydrolysis of ATP by this ATPase was negligible in the absence of magnesium or calcium ions. However, at millimolar concentrations of magnesium and calcium ions, the membrane-specific ATPase activity increased to about 560-600 U/mg, exhibiting two classes of ATP-hydrolysing sites, and site-site interactions. GTP, UTP, ITP, and CTP were also hydrolyzed by the membrane-specific ATPase. Oligomycin, ouabain, bafilomycin A(1), thapsigargin, omeprazole, ethacrynic acid and EDTA slightly affected membrane-specific ATPase activity while vanadate produced a 18% inhibition. The membrane-specific ATPase activity was insensitive to theophylline, but was inhibited 40% by levamisole. These data suggested that the membrane-specific ATPase activity present in osseous plate membranes, and alkaline phosphatase, were different proteins. (C) 1998 Elsevier B.V. B.V.

Proteoliposomes Harboring Alkaline Phosphatase and Nucleotide Pyrophosphatase as Matrix Vesicle Biomimetics

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Kinetic Analysis of Substrate Utilization by Native and TNAP-, NPP1-, or PHOSPHO1-Deficient Matrix Vesicles

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Contribution of matrix vesicles and alkaline phosphatase to ectopic bone formation

Endochondral calcification involves the participation of matrix vesicles (MVs), but it remains unclear whether calcification ectopically induced by implants of demineralized bone matrix also proceeds via MVs. Ectopic bone formation was induced by implanting rat demineralized diaphyseal bone matrix into the dorsal subcutaneous tissue of Wistar rats and was examined histologically and biochemically. Budding of MVs from chondrocytes was observed to serve as nucleation sites for mineralization during induced ectopic osteogenesis, presenting a diameter with Gaussian distribution with a median of 306 ± 103 nm. While the role of tissue-nonspecific alkaline phosphatase (TNAP) during mineralization involves hydrolysis of inorganic pyrophosphate (PPi), it is unclear how the microenvironment of MV may affect the ability of TNAP to hydrolyze the variety of substrates present at sites of mineralization. We show that the implants contain high levels of TNAP capable of hydrolyzing p-nitrophenylphosphate (pNPP), ATP and PPi. The catalytic properties of glycosyl phosphatidylinositol-anchored, polidocanol-solubilized and phosphatidylinositol-specific phospholipase C-released TNAP were compared using pNPP, ATP and PPi as substrates. While the enzymatic efficiency (k cat/Km) remained comparable between polidocanol-solubilized and membrane-bound TNAP for all three substrates, the k cat/Km for the phosphatidylinositol-specific phospholipase C-solubilized enzyme increased approximately 108-, 56-, and 556-fold for pNPP, ATP and PPi, respectively, compared to the membrane-bound enzyme. Our data are consistent with the involvement of MVs during ectopic calcification and also suggest that the location of TNAP on the membrane of MVs may play a role in determining substrate selectivity in this micro-compartment.

Dedução de Funções de Alívio para Priorização de Variantes de Chaveamento Adequadas à Eliminação de Sobrecargas

Eventually, violations of voltage limits at buses or admissible loadings of transmission lines and/or power transformers may occur by the power system operation. If violations are detected in the supervision process, corrective measures may be carried out in order to eliminate them or to reduce their intensity. Loading restriction is an extreme solution and should only be adopted as the last control action. Previous researches have shown that it is possible to control constraints in electrical systems by changing the network topology, using the technique named Corrective Switching, which requires no additional costs. In previous works, the proposed calculations for verifying the ability of a switching variant in eliminating an overload in a specific branch were based on network reduction or heuristic analysis. The purpose of this work is to develop analytical derivation of linear equations to estimate current changes in a specific branch (due to switching measures) by means of few calculations. For bus-bar coupling, derivations will be based on short-circuit theory and Relief Function methodology. For bus-bar splitting, a Relief Function will be derived based on a technique of equivalent circuit. Although systems of linear equations are used to substantiate deductions, its formal solution for each variant, in real time does not become necessary. A priority list of promising variants is then assigned for final check by an exact load flow calculation and a transient analysis using ATP Alternative Transient Program. At last, results obtained by simulation in networks with different features will be presented

Proposição de um modelo para análise de fluxo de carga, curto-circuito e simulação de relés de produção

The objective of the present work is develop a model to simulate electrical energy networks in transient and stead states, using the software ATP (Alternative Transient Program), able to be a way to join two distinct themes, present in classical methodology planning networks: short circuit analysis and load flow theory. Beyond that, using a tool for relay simulation, this paper intend to use the new developed model to investigate the influence of transient phenomenon in operation of protection relays, and calibrate the enterprise's protections relays. For testing the model, some relays, actually, installed at COSERN were used

Localização de faltas em linhas de transmissão de múltiplos terminais a partir de registros oscilográficos sincronizados via transformada wavelet

The electric energy is essential to the development of modern society and its increasing demand in recent years, effect from population and economic growth, becomes the companies more interested in the quality and continuity of supply, factors regulated by ANEEL (Agência Nacional de Energia Elétrica). These factors must be attended when a permanent fault occurs in the system, where the defect location that caused the power interruption should be identified quickly, which is not a simple assignment because the current systems complexity. An example of this occurs in multiple terminals transmission lines, which interconnect existing circuits to feed the demand. These transmission lines have been adopted as a feasible solution to suply loads of magnitudes that do not justify economically the construction of new substations. This paper presents a fault location algorithm for multiple terminals transmission lines - two and three terminals. The location method is based on the use of voltage and current fundamental phasors, as well as the representation of the line through its series impedance. The wavelet transform is an effective mathematical tool in signals analysis with discontinuities and, therefore, is used to synchronize voltage and current data. The Fourier transform is another tool used in this work for extract voltage and current fundamental phasors. Tests to validate the location algorithm applicability used data from faulty signals simulated in ATP (Alternative Transients Program) as well as real data obtained from oscillographic recorders installed on CHESF s lines.

Desenvolvimento de Modelos no Programa DigSILENT PowerFactoryTM para o Estudo de Estabilidade Transitória em Sistemas Elétricos de Potência com Aerogerador

This work presents a description of models development at DigSILENT PowerFactoryT M program for the transient stability study in power systems with wind turbine. The main goal is to make available means to use a dynamic simulation program in power systems, widely published, and utilize it as a tool that helps in programs results evaluations used for this intent. The process of simulations and analyses results starts after the models setting description phase. The results obtained by the DigSILENT PowerFactoryT M and ATP, program chosen to the validation also international recognized, are compared during this phase. The main tools and guide lines of PowerFactoryT M program use are presented here, directing these elements to the solution of the approached problem. For the simulation it is used a real system which it will be connected a wind farm. Two different technologies of wind turbines were implemented: doublyfed induction generator with frequency converter, connecting the rotor to the stator and to the grid, and synchronous wind generator with frequency converter, interconnecting the generator to the grid. Besides presenting the basic conceptions of dynamic simulation, it is described the implemented control strategies and models of turbine and converters. The stability of the wind turbine interconnected to grid is analyzed in many operational conditions, resultant of diverse kinds of disturbances

Desenvolvimento de modelos no programa digSILENT powerfactoryTM para o estudo de estabilidade transitória em sistemas elétricos de potência com aerogerador

This work presents a description of models development at DigSILENT PowerFactoryTM program for the transient stability study in power systems with wind turbine. The main goal is to make available means to use a dynamic simulation program in power systems, widely published, and utilize it as a tool that helps in programs results evaluations used for this intent. The process of simulations and analyses results starts after the models setting description phase. The results obtained by the DigSILENT PowerFactoryTM and ATP, program chosen to the validation also international recognized, are compared during this phase. The main tools and guide lines of PowerFactoryTM program use are presented here, directing these elements to the solution of the approached problem. For the simulation it is used a real system which it will be connected a wind farm. Two different technologies of wind turbines were implemented: doubly-fed induction generator with frequency converter, connecting the rotor to the stator and to the grid, and synchronous wind generator with frequency converter, interconnecting the generator to the grid. Besides presenting the basic conceptions of dynamic simulation, it is described the implemented control strategies and models of turbine and converters. The stability of the wind turbine interconnected to grid is analyzed in many operational conditions, resultant of diverse kinds of disturbances

Estimação de Fasores para Proteção de Sistemas Elétricos Baseada em Mínimos Quadrados e Morfologia Matemática

This work proposes a new technique for phasor estimation applied in microprocessor numerical relays for distance protection of transmission lines, based on the recursive least squares method and called least squares modified random walking. The phasor estimation methods have compromised their performance, mainly due to the DC exponential decaying component present in fault currents. In order to reduce the influence of the DC component, a Morphological Filter (FM) was added to the method of least squares and previously applied to the process of phasor estimation. The presented method is implemented in MATLABr and its performance is compared to one-cycle Fourier technique and conventional phasor estimation, which was also based on least squares algorithm. The methods based on least squares technique used for comparison with the proposed method were: forgetting factor recursive, covariance resetting and random walking. The techniques performance analysis were carried out by means of signals synthetic and signals provided of simulations on the Alternative Transient Program (ATP). When compared to other phasor estimation methods, the proposed method showed satisfactory results, when it comes to the estimation speed, the steady state oscillation and the overshoot. Then, the presented method performance was analyzed by means of variations in the fault parameters (resistance, distance, angle of incidence and type of fault). Through this study, the results did not showed significant variations in method performance. Besides, the apparent impedance trajectory and estimated distance of the fault were analysed, and the presented method showed better results in comparison to one-cycle Fourier algorithm

Coenzyme Q(10) and its effects in the treatment of neurodegenerative diseases

According to clinical and pre-clinical studies, oxidative stress and its consequences may be the cause or, at least, a contributing factor, to a large number of neurodegenerative diseases. These diseases include common and debilitating disorders, characterized by progressive and irreversible loss of neurons in specific regions of the brain. The most common neurodegenerative diseases are Parkinson's disease, Huntington's disease, Alzheimer's disease and amyotrophic lateral sclerosis. Coenzyme Q(10) (CoQ(10)) has been extensively studied since its discovery in 1957. It is a component of the electron transportation chain and participates in aerobic cellular respiration, generating energy in the form of adenosine triphosphate (ATP). The property of CoQ(10) to act as an antioxidant or a pro-oxidant, suggests that it also plays an important role in the modulation of redox cellular status under physiological and pathological conditions, also performing a role in the ageing process. In several animal models of neurodegenerative diseases, CoQ(10) has shown beneficial effects in reducing disease progression. However, further studies are needed to assess the outcome and effectiveness of CoQ(10) before exposing patients to unnecessary health risks at significant costs.

Differential gene expression analysis of Paracoccidioides brasiliensis during keratinocyte infection

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)