25 research outputs found

    Acompanhamento químico da vermicompostagem de lodo de esgoto doméstico

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    This research aims to monitor the humification process of domestic sewage sludge resulted from the vermicomposting, evaluating, also, the possibility of using the final product (vermicompost) in agricultural soils. The monitored chemical variables during the 90 days of vermicomposting were: humidity rate, organic matter content, nitrogen and phosphorus content, pathogenic organisms concentration, total organic carbon, acidity, CEC, C/N ratio, CEC/TOC ratio, and humic and fulvic acids content. The change in these variables during the vermicomposting process showed that this technique is effective for use in the maturation of the residue

    Chemical Differentiation of Domestic Sewage Sludge and Cattle Manure Stabilized by Microbioreators: Study by Pyrolysis Coupled to Gas Chromatography Coupled to Mass Spectroscopy

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    The aim of the present study was to evaluate the chemical alterations in substrates of vermicomposts from domestic sewage sludge and from cattle manure, besides its quality as an organic fertilizer. Elemental and infrared analysis and UV-Vis spectroscopy were used for their characterizations. Chemical characteristics determined in the vermicomposts indicate that they can be used as organic fertilizers, mainly with regard to organic matter content, pH, C/N ratio, and nitrogen and phosphorus levels. The main constituents of the humic substances extracted from the vermicomposts were obtained by pyrolysis coupled to gas chromatography-mass spectrometry (GC/MS), showing that these compounds are quite similar, despite being produced by domestic sewage sludges or by cattle manure

    Chemical Differentiation of Domestic Sewage Sludge and Cattle Manure Stabilized by Microbioreators: Study by Pyrolysis Coupled to Gas Chromatography Coupled to Mass Spectroscopy

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    The aim of the present study was to evaluate the chemical alterations in substrates of vermicomposts from domestic sewage sludge and from cattle manure, besides its quality as an organic fertilizer. Elemental and infrared analysis and UV-Vis spectroscopy were used for their characterizations. Chemical characteristics determined in the vermicomposts indicate that they can be used as organic fertilizers, mainly with regard to organic matter content, pH, C/N ratio, and nitrogen and phosphorus levels. The main constituents of the humic substances extracted from the vermicomposts were obtained by pyrolysis coupled to gas chromatography-mass spectrometry (GC/MS), showing that these compounds are quite similar, despite being produced by domestic sewage sludges or by cattle manure

    Processo de estabilização de resíduos orgùnicos: vermicompostagem versus compostagem

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    Two processes are used to stabilize organic wastes: composting and vermicomposting.There are no studies in the literature showing which process is most effective over the short term. In this study, 3 organic wastes were composted and vermicomposted for 90 days, and the parameters pH, effective cation exchange capacity, total organic carbon, total Kjeldahl nitrogen, Ptotal, E4/E6 ratio, hydrophobicity and aromaticity indexes were determined. In all experiments, vermicomposted materials showed higher stability, proving a superior tool for stabilization of these organic wastes

    Human mitochondrial Hsp70 (Mortalin): Shedding light on ATPase activity, interaction with adenosine nucleotides, solution structure and domain organization

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    The human mitochondrial Hsp70, also called mortalin, is of considerable importance for mitochondria biogenesis and the correct functioning of the cell machinery. In the mitochondrial matrix, mortalin acts in the importing and folding process of nucleus-encoded proteins. The in vivo deregulation of mortalin expression and/or function has been correlated with agerelated diseases and certain cancers due to its interaction with the p53 protein. In spite of its critical biological roles, structural and functional studies on mortalin are limited by its insoluble recombinant production. This study provides the first report of the production of folded and soluble recombinant mortalin when co-expressed with the human Hsp70-escort protein 1, but it is still likely prone to self-association. The monomeric fraction of mortalin presented a slightly elongated shape and basal ATPase activity that is higher than that of its cytoplasmic counterpart Hsp70-1A, suggesting that it was obtained in the functional state. Through small angle X-ray scattering, we assessed the low-resolution structural model of monomeric mortalin that is characterized by an elongated shape. This model adequately accommodated high resolution structures of Hsp70 domains indicating its quality. We also observed that mortalin interacts with adenosine nucleotides with high affinity. Thermally induced unfolding experiments indicated that mortalin is formed by at least two domains and that the transition is sensitive to the presence of adenosine nucleotides and that this process is dependent on the presence of Mg2+ ions. Interestingly, the thermal-induced unfolding assays of mortalin suggested the presence of an aggregation/association event, which was not observed for human Hsp70-1A, and this finding may explain its natural tendency for in vivo aggregation. Our study may contribute to the structural understanding of mortalin as well as to contribute for its recombinant production for antitumor compound screenings.The human mitochondrial Hsp70, also called mortalin, is of considerable importance for mitochondria biogenesis and the correct functioning of the cell machinery. In the mitochondrial matrix, mortalin acts in the importing and folding process of nucleus-en101FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOSEM INFORMAÇÃOSEM INFORMAÇÃOWadhwa, R., Kaul, S.C., Ikawa, Y., Sugimoto, Y., Identification of a novel member of mouse hsp70 family. Its association with cellular mortal phenotype (1993) J Biol Chem, 268, pp. 6615-6621. , PMID: 8454632Bhattacharyya, T., Karnezis, A.N., Murphy, S.P., Hoang, T., Freeman, B.C., Cloning and subcellular localization of human mitochondrial hsp70 (1995) J Biol Chem, 270, pp. 1705-1710. , PMID: 7829505Domanico, S.Z., DeNagel, D.C., Dahlseid, J.N., Green, J.M., Pierce, S.K., Cloning of the gene encoding peptide-binding protein 74 shows that it is a new member of the heat shock protein 70 family (1993) Mol Cell Biol, 13, pp. 3598-3610. , PMID: 7684501Londono, C., Osorio, C., Gama, V., Alzate, O., Mortalin, Apoptosis, and Neurodegeneration (2012) Biomolecules, 2, pp. 143-164. , PMID: 24970131Wadhwa, R., Sugihara, T., Yoshida, A., Nomura, H., Reddel, R.R., Selective toxicity of MKT-077 to cancer cells is mediated by its binding to the hsp70 family protein mot-2 and reactivation of p53 function (2000) Cancer Res, 60, pp. 6818-6821. , PMID: 11156371Wadhwa, R., Yaguchi, T., Hasan, K., Mitsui, Y., Reddel, R.R., Hsp70 family member, mot-2/ mthsp70/GRP75, binds to the cytoplasmic sequestration domain of the p53 protein (2002) Exp Cell Res, 274, pp. 246-253. , PMID: 11900485Kaul, S.C., Deocaris, C.C., Wadhwa, R., Three faces of mortalin: A housekeeper, guardian and killer (2007) Exp Gerontol, 42, pp. 263-274. , PMID: 17188442Fan, A.C.Y., Young, J.C., Function of cytosolic chaperones in Tom70-mediated mitochondrial import (2011) Prot Pept Lett, 18, pp. 122-131Dolezal, P., Likic, V., Tachezy, J., Lithgow, T., Evolution of the molecular machines for protein import into mitochondria (2006) Science, 313, pp. 314-318. , PMID: 16857931Mokranjac, D., Neupert, W., Thirty years of protein translocation into mitochondria: Unexpectedly complex and still puzzling (2009) BBA-Mol Cell Res, 1793, pp. 33-41Bohnert, M., Pfanner, N., Van Der Laan, M., A dynamic machinery for import of mitochondrial precursor proteins (2007) Febs Lett, 581, pp. 2802-2810. , PMID: 17376437Kaul, S.C., Reddel, R.R., Sugihara, T., Mitsui, Y., Wadhwa, R., Inactivation of p53 and life span extension of human diploid fibroblasts by mot-2 (2000) Febs Lett, 474, pp. 159-164Wadhwa, R., Takano, S., Kaur, K., Deocaris, C.C., Pereira-Smith, O.M., Upregulation of mortalin/ mthsp70/Grp75 contributes to human carcinogenesis (2006) Inter J Cancer, 118, pp. 2973-2980Qu, M., Zhou, Z., Xu, S., Chen, C., Yu, Z., Mortalin overexpression attenuates beta-amyloid-induced neurotoxicity in SH-SY5Y cells (2011) Brain Res, 1368, pp. 336-345. , PMID: 20974113Liu, Y., Liu, W., Song, X.D., Zuo, J., Effect of GRP75/mthsp70/PBP74/mortalin overexpression on intracellular ATP level, mitochondrial membrane potential and ROS accumulation following glucose deprivation in PC12 cells (2005) Mol Cell Biochem, 268, pp. 45-51. , PMID: 15724436Jin, J.H., Hulette, C., Wang, Y., Zhang, T., Pan, C., Proteomic identification of a stress protein, mortalin/mthsp70/GRP75-Relevance to Parkinson disease (2006) Mol Cell Proteomics, 5, pp. 1193-1204. , PMID: 16565515Xu, L., Voloboueva, L.A., Ouyang, Y., Emery, J.F., Giffard, R.G., Overexpression of mitochondrial Hsp70/Hsp75 in rat brain protects mitochondria, reduces oxidative stress, and protects from focal ischemia (2008) J Cereb Blood Flow Metab, 29, pp. 365-374. , PMID: 18985056Wadhwa, R., Takano, S., Kaur, K., Aida, S., Yaguchi, T., Identification and characterization of molecular interactions between mortalin/mtHsp70 and HSP60 (2005) Biochem J, 391, pp. 185-190. , PMID: 15957980Rehling, P., Brandner, K., Pfanner, N., Mitochondrial import and the twin-pore translocase (2004) Nat Rev Mol Cell Biol, 5, pp. 519-530. , PMID: 15232570Wegele, H., Muller, L., Buchner, J., Hsp70 and Hsp90-a relay team for protein folding (2004) Rev Physiol Biochem Pharmacol, 151, pp. 1-44. , PMID: 14740253Soti, C., Nagy, E., Giricz, Z., Vigh, L., Csermely, P., Heat shock proteins as emerging therapeutic targets (2005) Br J Pharmacol, 146, pp. 769-780. , PMID: 16170327Da Silva, K.P., Borges, J.C., The molecular chaperone Hsp70 family members function by a bidirectional heterotrophic allosteric mechanism (2011) Protein Pept Lett, 18, pp. 132-142. , PMID: 21121894Broadley, S.A., Hartl, F.U., The role of molecular chaperones in human misfolding diseases (2009) FEBS Lett, 583, pp. 2647-2653. , PMID: 19393652Deocaris, C.C., Kaul, S.C., Wadhwa, R., Mortalin's machinery (2012) Mortalin Biology: Life, Stress and Death., pp. 21-30. , Sunil C. Kaul, Renu Wadhwa SpringerWadhwa, R., Kaul, S.C., Sugimoto, Y., Mitsui, Y., Induction of cellular senescence by transfection of cytosolic mortalin Cdna in Nih-3T3-Cells (1993) J Biol Chem, 268, pp. 22239-22242. , PMID: 7693662Deocaris, C.C., Kaul, S.C., Wadhwa, R., Mortalin's machinery (2012) Mortalin Biology: Life, Stress and Death, pp. 21-30. , Kaul SC, Wadhwa R, editors New York: Dordrecht: SpringerWadhwa, R., Kaul, S.C., Mitsui, Y., Sugimoto, Y., Differential subcellular-distribution of mortalin in mortal and immortal mouse and human fibroblasts (1993) Exp Cell Res, 207, pp. 442-448. , PMID: 8344392Ran, Q.T., Wadhwa, R., Kawai, R., Kaul, S.C., Sifers, R.N., Extramitochondrial localization of mortalin/ mthsp70/PBP74/GRP75 (2000) Biochem Biophys Res Comm, 275, pp. 174-179. , PMID: 10944461Deocaris, C.C., Widodo, N., Shrestha, B.G., Kaur, K., Ohtaka, M., Mortalin sensitizes human cancer cells to MKT-077-induced senescence (2007) Cancer Lett, 252, pp. 259-269. , PMID: 17306926Szklarz, L.K.S., Guiard, B., Rissler, M., Wiedemann, N., Kozjak, V., Inactivation of the mitochondrial heat shock protein Zim17 leads to aggregation of matrix Hsp70s followed by plelotropic effects on morphology and protein biogenesis (2005) J Mol Biol, 351, pp. 206-218Sichting, M., Mokranjac, D., Azem, A., Neupert, W., Hell, K., Maintenance of structure and function of mitochondrial Hsp70 chaperones requires the chaperone Hep1 (2005) EMBO J, 24, pp. 1046-1056. , PMID: 15719019Zhai, P., Stanworth, C., Liu, S., Silberg, J.J., The human escort protein hep binds to the ATPase domain of mitochondrial Hsp70 and regulates ATP hydrolysis (2008) J Biol Chem, 283, pp. 26098-26106. , PMID: 18632665Luo, W.I., Dizin, E., Yoon, T., Cowan, J.A., Kinetic and structural characterization of human mortalin (2010) Protein Expr Purif, 72, pp. 75-81. , PMID: 20152901Dores-Silva, P.R., Minari, K., Ramos, C.H.I., Barbosa, L.R.S., Borges, J.C., Structural and stability studies of the human mtHsp70-escort protein 1: An essential mortalin co-chaperone (2013) Int J Biol Macromol, 56, pp. 140-148. , PMID: 23462535Vu, M.T., Zhai, P., Lee, J., Guerra, C., Liu, S., The DNLZ/HEP zinc-binding subdomain is critical for regulation of the mitochondrial chaperone HSPA9 (2012) Protein Sci, 21, pp. 258-267. , 10.1002/pro.2012 PMID: 22162012Zhai, P., Vu, M.T., Hoff, K.G., Silberg, J.J., A conserved histidine in human DNLZ/HEP is required for stimulation of HSPA9 ATPase activity (2011) Biochem Bioph Res Co, 408, pp. 589-594Borges, J.C., Ramos, C.H.I., Spectroscopic and thermodynamic measurements of nucleotide-induced changes in the human 70-kDa heat shock cognate protein (2006) Arch Biochem Biophys, 452, pp. 46-54. , PMID: 16806043Bohm, G., Muhr, R., Jaenicke, R., Quantitative-Analysis of protein far Uv circular-dichroism spectra by neural networks (1992) Protein Eng, 5, pp. 191-195. , PMID: 1409538Lebowitz, J., Lewis, M.S., Schuck, P., Modern analytical ultracentrifugation in protein science: A tutorial review (2002) Protein Sci, 11, pp. 2067-2079. , PMID: 12192063Schuck, P., Perugini, M.A., Gonzales, N.R., Howlett, G.J., Schubert, D., Size-distribution analysis of proteins by analytical ultracentrifugation: Strategies and application to model systems (2002) Biophys J, 82, pp. 1096-1111. , PMID: 11806949Borges, J.C., Ramos, C.H.I., Analysis of molecular targets of mycobacterium tuberculosis by analytical ultracentrifugation (2011) Curr Med Chem, 18, pp. 1276-1285. , PMID: 21366535Orthaber, D., Bergmann, A., Glatter, O., SAXS experiments on absolute scale with Kratky systems using water as a secondary standard (2000) J Appl Cryst, 33, pp. 218-225Svergun, D.I., Restoring low resolution structure of biological macromolecules from solution scattering using simulated annealing (1999) Biophys J, 76, pp. 2879-2886. , PMID: 10354416Volkov, V.V., Svergun, D.I., Uniqueness of ab initio shape determination in small-angle scattering (2003) J Appl Cryst, 36, pp. 860-864De La Torre, J.G., Huertas, M.L., Carrasco, B., Calculation of hydrodynamic properties of globular proteins from their atomic-level structure (2000) Biophys J, 78, pp. 719-730Kityk, R., Kopp, J., Sinning, I., Mayer, M.P., Structure and dynamics of the ATP-Bound open conformation of Hsp70 chaperones (2012) Mol Cell, 48, pp. 863-874. , PMID: 23123194Bertelsen, E.B., Chang, L., Gestwicki, J.E., Zuiderweg, E.R.P., Solution conformation of wild-type E. Coli Hsp70 (DnaK) chaperone complexed with ADP and substrate (2009) Proc Natl Acad Sci U S A, 106, pp. 8471-8476. , PMID: 19439666Spinozzi, F., Ferrero, C., Ortore, M.G., De Maria Antolinos, A., Mariani, P., GENFIT: Software for the analysis of small-angle X-ray and neutron scattering data of macromolecules in solution (2014) J Appl Cryst, 47, pp. 1132-1139Ortore, M.G., Spinozzi, F., Mariani, P., Paciaroni, A., Barbosa, L.R.S., Combining structure and dynamics: Non-denaturing high-pressure effect on lysozyme in solution (2009) J R Soc Interface, 6, pp. S619-S634. , PMID: 19570795Silva, K.P., Seraphim, T.V., Borges, J.C., Structural and functional studies of Leishmania braziliensis Hsp90 (2013) BBA-Proteins Proteom, 1834, pp. 351-361Borges, J.C., Ramos, C.H.I., Characterization of nucleotide-induced changes on the quaternary structure of human 70 kDa heat shock protein Hsp70.1 by analytical ultracentrifugation (2009) BMB Rep, 42, pp. 166-171. , PMID: 19336004Palleros, D.R., Welch, W.J., Fink, A.L., Interaction of hsp70 with unfolded proteins: Effects of temperature and nucleotides on the kinetics of binding (1991) Proc Natl Acad Sci U S A, 88, pp. 5719-5723. , PMID: 1829527Montgomery, D.L., Morimoto, R.I., Gierasch, L.M., Mutations in the substrate binding domain of the Escherichia coli 70 kDa molecular chaperone, DnaK, which alter substrate affinity or interdomain coupling (1999) J Mol Biol, 286, pp. 915-932. , PMID: 10024459Palleros, D.R., Reid, K.L., Shi, L., Fink, A.L., DnaK ATPase activity revisited (1993) Febs Letters, 336, pp. 124-128. , PMID: 8262193Kozin, M.B., Svergun, D.I., Automated matching of high- and low-resolution structural models (2001) J Appl Cryst, 34, pp. 33-41Barbosa, L.R.S., Spinozzi, F., Mariani, P., Itri, R., (2013) Small-angle X-ray Scattering Applied to Proteins in Solution., pp. 49-72. , Proteins in Solution and at Interfaces. 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CHIR and LRSB thank FAPESP and CNPq for financial support. We also thank t

    Low resolution structural characterization of the Hsp70-interacting protein - Hip - from Leishmania braziliensis emphasizes its high asymmetry

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    The Hsp70 is an essential molecular chaperone in protein metabolism since it acts as a pivot with other molecular chaperone families. Several co-chaperones act as regulators of the Hsp70 action cycle, as for instance Hip (Hsp70-interacting protein). Hip is a tetratricopeptide repeat protein (TPR) that interacts with the ATPase domain in the Hsp70-ADP state, stabilizing it and preventing substrate dissociation. Molecular chaperones from protozoans, which can cause some neglected diseases, are poorly studied in terms of structure and function. Here, we investigated the structural features of Hip from the protozoa Leishmania braziliensis (LbHip), one of the causative agents of the leishmaniasis disease. LbHip was heterologously expressed and purified in the folded state, as attested by circular dichroism and intrinsic fluorescence emission techniques. LbHip forms an elongated dimer, as observed by analytical gel filtration chromatography, analytical ultracentrifugation and small angle X-ray scattering (SAXS). With the SAXS data a low resolution model was reconstructed, which shed light on the structure of this protein, emphasizing its elongated shape and suggesting its domain organization. We also investigated the chemical-induced unfolding behavior of LbHip and two transitions were observed. The first transition was related to the unfolding of the TPR domain of each protomer and the second transition of the dimer dissociation. Altogether. LbHip presents a similar structure to mammalian Hip, despite their low level of conservation, suggesting that this class of eukaryotic protein may use a similar mechanism of action. (C) 2012 Elsevier Inc. All rights reserved.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)FAPESP (Fundacao de Amparo a Pesquisa do Estado de Sao Paulo) [2007/05001-4]CNPq (Conselho Nacional de Pesquisa e Desenvolvimento)Conselho Nacional de Pesquisa e Desenvolvimento (CNPq)CAPES (Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior)Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES

    Summary of the Tm transitions determined to mortalin by CD<sub>222 nm</sub> in the presence of adenosine nucleotides and/or Mg<sup>2+</sup>.

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    <p>Values obtained by averaging 4 independent preparations.</p><p>Summary of the Tm transitions determined to mortalin by CD<sub>222 nm</sub> in the presence of adenosine nucleotides and/or Mg<sup>2+</sup>.</p

    Kinetic constants determined for human mortalin and human Hsp70–1A compared with those of homologous Hsp70.

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    <p>Comparing the k<sub>cat</sub> to other members of Hsp70 family we notice that the values are too close of each other, Sadis & Hightower [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117170#pone.0117170.ref067" target="_blank">67</a>] found a k<sub>cat</sub> of 0.15 min<sup>-1</sup> for bovine brain Hsc70, while the recombinant rat Hsc70 presented 0.12 min<sup>-1</sup> for the ATPase activity.</p><p>Kinetic constants determined for human mortalin and human Hsp70–1A compared with those of homologous Hsp70.</p
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