Cytoplasmatic domain of Na,K-ATPase α-subunit is responsible for the aggregation of the enzyme in proteoliposomes

We studied the thermal dependence of amide I′ infrared absorption and fluorescence emission of Trp residues in the Na,K-ATPase of rabbit kidney. We studied the whole enzyme solubilized with detergent, the whole enzyme reconstituted in proteoliposomes and the protein fraction that remained in the lipid membrane after the trypsin digestion of the proteoliposomes. Cooperative unfolding and aggregation with increasing temperature were observed in the whole protein, whether solubilized or reconstituted, but not in the fraction remaining after trypsinization. The protein influenced the physical state of the lipid, decreasing the temperature of the gel to liquid-crystalline phase transition and the degree of cooperativity. This study provides new information for the understanding of the processes controlling the association mechanisms that are important for enzyme function in natural membranes. © 2009 Elsevier B.V. All rights reserved.Fil: Rigos, Carolina Fortes. Universidade de Sao Paulo; BrasilFil: de Lima Santos, Hérica. Universidade Federal de Sao Joao Del-rei; BrasilFil: Yoneda, Juliana Sakamoto. Universidade de Sao Paulo; BrasilFil: Montich, Guillermo Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Maggio, Bruno. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Ciancaglini, Pietro. Universidade de Sao Paulo; Brasi

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

During the process of endochondral bone formation, chondrocytes and osteoblasts mineralize their extracellular matrix by promoting the formation of hydroxyapatite seed crystals in the sheltered interior of membrane-limited matrix vesicles (MVs). Here, we have studied phosphosubstrate catalysis by osteoblast-derived MVs at physiologic pH, analyzing the hydrolysis of ATP, ADP, and PPi by isolated wild-type (WT) as well as TNAP-, NPP1- and PHOSPHO1-deficient MVs. Comparison of the catalytic efficiencies identified ATP as the main substrate hydrolyzed by WT MVs. The lack of TNAP had the most pronounced effect on the hydrolysis of all physiologic substrates. The lack of PHOSPHO1 affected ATP hydrolysis via a secondary reduction in the levels of TNAP in PHOSPHO1-deficient MVs. The lack of NPP1 did not significantly affect the kinetic parameters of hydrolysis when compared with WT MVs for any of the substrates. We conclude that TNAP is the enzyme that hydrolyzes both ATP and PPi in the MV compartment. NPP1 does not have a major role in PPi generation from ATP at the level of MVs, in contrast to its accepted role on the surface of the osteoblasts and chondrocytes, but rather acts as a phosphatase in the absence of TNAP. © 2010 American Society for Bone and Mineral Research

The kinetic behavior of dehydrogenase enzymes in solution and immobilized onto nanostructured carbon platforms

This paper describes the kinetic behavior of alcohol (ADH) and aldehyde (AldDH) dehydrogenases in solution and immobilized onto carbon platform via polyamidoamine (PAMAM) dendrimers. All the kinetic constants achieved for soluble ADH and AldDH are in agreement with literature data. The influence of pH and temperature was evaluated. Results showed that physiological conditions and ambient temperature can satisfactorily be applied to systems containing dehydrogenase enzymes, so as to ensure an environment where both ADH and AldDH display good activity. It is noteworthy that the affinity between both ADH and AldDH and their substrates and coenzyme is retained after the immobilization process. Investigation of the influence of the storage time demonstrated that there was no appreciable reduction in enzymatic activity for 50 days. Results showed that the PAMAM dendrimers provide a good environment for immobilization of dehydrogenase enzymes and that the affinity observed between the enzymes and their substrates and coenzymes seems to be retained, despite the considerable loss of enzymatic activity after immobilization. Furthermore, the anchoring methodology employed herein, namely layer-by-layer (LbL), required very low catalyst consumption.FAPESPCNPqCAPE

Amazonian biodiversity: a view of drug development for Leishmaniasis and malaria

Chemotherapy is the only validated therapy for the treatment for the neglected diseases leishmaniasis and malaria. However, the emergence of drug resistance, collateral effects and long-term treatment encourage the development of new and more efficient drugs. The Amazon tropical forest includes the richest areas of biodiversity in the world, including a great number of microbes, plant and animal species that produce a source of interesting biologically active molecules. Several of these molecules, obtained from plant extracts and frog venom have leishmanicidal and plasmodicidal activity, highlighting the potential of this biodiversity for the development of new drugs. In research, modern approaches in new drug development are carried out using combinatorial chemistry, high-throughput screening, bioinformatics, molecular interaction, crystallography and dynamic studies of cellular and systemic toxicity. In Brazil, these techniques are mainly present in only a few academic groups with no efficient connection to industry. The problem associated with over-regulation for accessing the biological material in restricted areas, local populations and indigenous areas places major barriers in the path of research and development of new drugs. Thus, the association of academic research groups in Brazil, encouraged and supported by government and industry, is essential to overcome these major barriers related to the development of new products for treatment of neglected diseases from Amazonian biodiversity in future years.A quimioterapia é o único procedimento farmacológico validado para a terapêutica da leishmaniose e da malária, consideradas doenças negligenciadas para o desenvolvimento de fármacos pelas indústrias farmacêuticas. Com a não renovação medicamentosa, o surgimento de resistência, os efeitos colaterais e o longo período de tratamento indicam a necessidade do desenvolvimento de novos e mais eficientes fármacos. A Floresta Amazônica é a região com a maior biodiversidade do planeta, com uma riqueza de animais e plantas produtoras de moléculas com atividades biológicas relevantes para o desenvolvimento e exploração biotecnológica. Algumas destas moléculas, obtidas de extratos vegetais e de venenos de anuros, apresentam atividade leishmanicida e plasmodicida, o que demonstra o potencial desta biodiversidade para a investigação de novas drogas. A moderna abordagem na pesquisa de novos fármacos envolve a associação de química combinatória, high-throughput screening, bioinformática, interação molecular, cristalografia e o estudo dinâmico de toxicidade sistêmica e celular, que atualmente no Brasil, estão distribuídas em poucos grupos acadêmicos sem a devida associação industrial. Esta deficiência, agregada ao excesso de regulamentação para o acesso ao material biológico, sobretudo, proveniente de unidades de conservação, populações tradicionais e nações indígenas, é um importante entrave para o desenvolvimento deste tipo de pesquisa. A associação de grupos de pesquisa do Brasil, estimulados por políticas governamentais de financiamento acadêmico e industrial, são essenciais para a superação destas dificuldades, de forma que nos próximos anos possam surgir novos produtos para terapia de doenças negligenciadas oriundas da biodiversidade amazônica

The biochemistry of mineralizing extracellular vesicles. Part I: The role of phosphatases

In this chapter, we will review some of the information regarding the functional significance of the inorganic phosphate (Pi)/pyrophosphate (PPi) ratio for physiological mineralization of hard tissues. We will recount the structure and function of the phosphatases involved in the regulation of this ratio: Tissue-nonspecific alkaline phosphatase (TNAP); Nucleotide Pyrophosphatases/Phosphodiesterase 1 (ENPP1); Na,K-ATPase; Nucleoside triphosphate diphosphohydrolase 1 (CD39); ecto-5′-nucleotidase (CD73) and orphan phosphatase 1 (PHOSPHO1); and how this knowledge has guided the development of protein therapeutics and of small molecule inhibitors to affect the Pi/PPi ratio in pathological conditions ranging from soft bones to ectopic calcification disorders

Do Media Extracellular Vesicles and Extracellular Vesicles Bound to the Extracellular Matrix Represent Distinct Types of Vesicles?

Mineralization-competent cells, including hypertrophic chondrocytes, mature osteoblasts, and osteogenic-differentiated smooth muscle cells secrete media extracellular vesicles (media vesicles) and extracellular vesicles bound to the extracellular matrix (matrix vesicles). Media vesicles are purified directly from the extracellular medium. On the other hand, matrix vesicles are purified after discarding the extracellular medium and subjecting the cells embedded in the extracellular matrix or bone or cartilage tissues to an enzymatic treatment. Several pieces of experimental evidence indicated that matrix vesicles and media vesicles isolated from the same types of mineralizing cells have distinct lipid and protein composition as well as functions. These findings support the view that matrix vesicles and media vesicles released by mineralizing cells have different functions in mineralized tissues due to their location, which is anchored to the extracellular matrix versus free-floating

Quantitative atomic force microscopy provides new insight into matrix vesicle mineralization

International audienc

Biosensors for efficient diagnosis of Leishmaniasis: innovations in bioanalytics for a neglected disease

The need for reliable, fast diagnostics is closely linked to the need for safe, effective treatment of the so-called “neglected” diseases. The list of diseases with no field-adapted diagnostic tools includes leishmaniasis, shigella, typhoid, and bacterial meningitis. Leishmaniasis, in particular, is a parasitic disease caused by Leishmania spp. transmitted by infected phlebotomine sandfly, which remains a public health concern in developing countries with ca. 12 million people infected and 350 million at risk of infection. Despite several attempts, methods for diagnosis are still noneffective, especially with regard to specificity due to false positives with Chagas’ disease caused by Trypanosoma cruzi. Accepted golden standards for detecting leishmaniasis involve isolation of parasites either microscopically, or by culture, and in both methods specimens are obtained by invasive means. Here, we show that efficient distinction between cutaneous leishmaniasis and Chagas’ disease can be obtained with a low-cost biosensor system made with nanostructured films containing specific Leishmania amazonensis and T. cruzi antigens and employing impedance spectroscopy as the detection method. This unprecedented selectivity was afforded by antigen−antibody molecular recognition processes inherent in the detection with the immobilized antigens, and by statistically correlating the electrical impedance data, which allowed distinction between real samples that tested positive for Chagas’ disease and leishmaniasis. Distinction could be made of blood serum samples containing 10−5 mg/mL of the antibody solution in a few minutes. The methods used here are generic and can be extended to any type of biosensor, which is important for an effective diagnosis of many other diseases.FAPESPCNPqCAPE