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191.
Adalberto Vieyra 《Bioscience reports》1996,16(2):115-127
Organic solutes such as urea, methylamines, polyols and amino acid can accumulate in the cytoplasm of cells to compensate for hyperosmotic conditions in the external medium. Whereas urea is considered to be typical of solutes that destabilize structure and function of proteins, methylamines, polyols and some amino acids appear to have the opposite effect, and can also compensate for the perturbing effects of urea. These effects have been extensively analyzed for a variety of proteins in terms of global changes in enzyme structure and acceleration or inhibition of overall reaction rates. Here the influence of these solutes on sarcoplasmic reticulum and plasma membrane (Ca2+ + Mg2+)ATPases is reviewed. The focus is on the changes induced by perturbing and stabilizing solutes at specific steps of the catalytic cycles of these enzymes, which can run forward (leading to ATP hydrolysis) and backward (leading to ATP synthesis). Structural changes promoted by osmolytes are correlated with functional changes, especially those that are related to energy coupling.This review is dedicated to Prof. Carlos Chagas Filho, founder of the Institute of Biophysics, on the occasion of its 50th anniversary. 相似文献
192.
Gessi S Merighi S Varani K Cattabriga E Benini A Mirandola P Leung E Mac Lennan S Feo C Baraldi S Borea PA 《Journal of cellular physiology》2007,211(3):826-836
Adenosine may affect several pathophysiological processes, including cellular proliferation, through interaction with A(1), A(2A), A(2B), and A(3) receptors. In this study we characterized adenosine receptors in human colon cancer tissues and in colon cancer cell lines Caco2, DLD1, HT29. mRNA of all adenosine subtypes was detected in cancer tissues and cell lines. At a protein levels low amount of A(1), A(2A), and A(2B) receptors were detected, whilst the A(3) was the most abundant subtype in both cancer tissues and cells, with a pharmacological profile typical of the A(3) subtype. All the receptors were coupled to stimulation/inhibition of adenylyl-cyclase in cancer cells, with the exception of A(1) subtype. Adenosine increased cell proliferation with an EC(50) of 3-12 microM in cancer cells. This effect was not essentially reduced by adenosine receptor antagonists. However dypiridamol, an adenosine transport inhibitor, increased the stimulatory effect induced by adenosine, suggesting an action at the cell surface. Addition of adenosine deaminase makes the A(3) agonist 2-chloro-N6-(3-iodobenzyl)-N-methyl-5'-carbamoyladenosine (Cl-IB-MECA) able to stimulate cell proliferation with an EC(50) of 0.5-0.9 nM in cancer cells, suggesting a tonic proliferative effect induced by endogenous adenosine. This effect was antagonized by 5-N-(4-methoxyphenyl-carbamoyl)amino-8-propyl-2(2furyl)-pyrazolo-[4,3e]-1,2,4-triazolo [1,5-c] pyrimidine (MRE 3008F20) 10 nM. Cl-IB-MECA-stimulated cell proliferation involved extracellular-signal-regulated-kinases (ERK1/2) pathway, as demonstrated by reduction of proliferation with 1,4-diamino-2,3-dicyano-1,4-bis-[2-amino-phenylthio]-butadiene (U0126) and by ERK1/2 phosphorylation. In conclusion this study indicates for the first time that in colon cancer cell lines endogenous adenosine, through the interaction with A(3) receptors, mediates a tonic proliferative effect. 相似文献
193.
Gessi S Varani K Merighi S Fogli E Sacchetto V Benini A Leung E Mac-Lennan S Borea PA 《Purinergic signalling》2007,3(1-2):109-116
Adenosine is a potent extracellular messenger that is produced in high concentrations under metabolically unfavourable conditions.
Tissue hypoxia, consequent to a compromised cellular energy status, is followed by the enhanced breakdown of ATP leading to
the release of adenosine. Through the interaction with A2 and A3 membrane receptors, adenosine is devoted to the restoration of tissue homeostasis, acting as a retaliatory metabolite. Several
aspects of the immune response have to be taken into consideration and even though in general it is very important to dampen
inflammation, in some circumstances, such as the case of cancer, it is also necessary to increase the activity of immune cells
against pathogens. Therefore, adenosine receptors that are defined as “sensors” of metabolic changes in the local tissue environment
may be very important targets for modulation of immune responses and drugs devoted to regulating the adenosinergic system
are promising in different clinical situations. 相似文献
194.
Mixtures containing lysozyme, LYSO, and a fully fluorinated surfactant, lithium perfluorononanoate, LiPFN, were investigated in a wide range of concentrations and mole ratios. To ensure consistency to the data, a comparison was made, when possible, with the more conventional SDS as surfactant. Molecular solutions, precipitates, and micellar phases have been observed. The region of existence for each phase depends on the LiPFN/LYSO mole ratios, r, and was determined by different experimental methods. Optical absorbance, CD, 19F NMR, viscosity, electrical conductivity, and dielectric relaxation methods were used. Some methods give information on the protein conformation, others on the state of the surfactant or on the collective system properties, respectively. Addition of LiPFN gives rise to a solution, a poly phase dispersion (at low surfactant to protein ratios) and to a micelle-mediated redissolution of the precipitates. Concomitant to the above macroscopic properties, peculiar effects in the state of LYSO are observed. Low amounts of surfactant reduce significantly the amount of alpha-helix in favor of the beta-sheet conformation of the protein. The former is almost completely regained once micelle-assisted redissolution of the complex occurs. The tertiary structure of the protein, conversely, is lost at low surfactant content and never recovered. Such evidence suggests the occurrence of a molten globule conformation for LYSO in micellar media. 相似文献
195.
196.
Heterologous expression and purification of active L‐asparaginase I of Saccharomyces cerevisiae in Escherichia coli host 下载免费PDF全文
João H. P. M. Santos Iris M. Costa João V. D. Molino Mariana S. M. Leite Marcela V. Pimenta João A. P. Coutinho Adalberto Pessoa Jr Sónia P. M. Ventura Gisele Monteiro 《Biotechnology progress》2017,33(2):416-424
l ‐asparaginase (ASNase) is a biopharmaceutical widely used to treat child leukemia. However, it presents some side effects, and in order to provide an alternative biopharmaceutical, in this work, the genes encoding ASNase from Saccharomyces cerevisiae (Sc_ASNaseI and Sc_ASNaseII) were cloned in the prokaryotic expression system Escherichia coli. In the 93 different expression conditions tested, the Sc_ASNaseII protein was always obtained as an insoluble and inactive form. However, the Sc_ASNaseI (His)6‐tagged recombinant protein was produced in large amounts in the soluble fraction of the protein extract. Affinity chromatography was performed on a Fast Protein Liquid Chromatography (FPLC) system using Ni2+‐charged, HiTrap Immobilized Metal ion Affinity Chromatography (IMAC) FF in order to purify active Sc_ASNaseI recombinant protein. The results suggest that the strategy for the expression and purification of this potential new biopharmaceutical protein with lower side effects was efficient since high amounts of soluble Sc_ASNaseI with high specific activity (110.1 ± 0.3 IU mg?1) were obtained. In addition, the use of FPLC‐IMAC proved to be an efficient tool in the purification of this enzyme, since a good recovery (40.50 ± 0.01%) was achieved with a purification factor of 17‐fold. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:416–424, 2017 相似文献
197.
Letícia C. de Lencastre Novaes Priscila G. Mazzola Adalberto Pessoa Jr. Thereza C. Vessoni Penna 《Biotechnology progress》2010,26(1):252-256
Green fluorescent protein (GFP) shows remarkable structural stability and high fluorescence; its stability can be directly related to its fluorescence output, among other characteristics. GFP is stable under increasing temperatures, and its thermal denaturation is highly reproducible. Some polymers, such as polyethylene glycol, are often used as modifiers of characteristics of biological macromolecules, to improve the biochemical activity and stability of proteins or drug bioavailability. The aim of this study was to evaluate the thermal stability of GFP in the presence of different PEG molar weights at several concentrations and exposed to constant temperatures, in a range of 70–95°C. Thermal stability was expressed in decimal reduction time. It was observed that the D‐values obtained were almost constant for temperatures of 85, 90, and 95°C, despite the PEG concentration or molar weight studied. Even though PEG can stabilize proteins, only at 75°C, PEG 600 and 4,000 g/mol stabilized GFP. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 相似文献
198.
199.
The ubiquitous bacterial second messenger c-di-GMP regulates the expression of various virulence determinants in a wide range of bacterial pathogens. Several studies have suggested that proteins with a PilZ domain function as c-di-GMP receptors. We have identified in the Pseudomonas aeruginosa genome eight genes encoding for PilZ orhologues and demonstrated binding of c-di-GMP to all but one of these proteins in a direct ligand binding assay. One protein with the PilZ domain, Alg44, is involved in biosynthesis of the extracellular polysaccharide alginate. We have shown that increasing c-di-GMP levels by overexpression of highly active diguanylate cyclases, or hydrolysis of c-di-GMP by phosphodiesterases, enhanced or reduced formation of alginate in mucoid strains, respectively. We have engineered substitutions in several conserved residues of the PilZ domain of Alg44 determined that they resulted in simultaneous loss of c-di-GMP binding and the ability to support production of alginate in P. aeruginosa. A 6xHis-tagged Alg44 fusion was also shown to localize in the membrane fraction of P. aeruginosa independently from its ability to bind c-di-GMP. Alg44 appears to be an essential component of the alginate biosynthetic apparatus, where, following binding of c-di-GMP, it controls polymerization or transport of the polysaccharide. 相似文献
200.
Neil C Thomson Adalberto S Rubin Robert M Niven Paul A Corris Hans Christian Siersted Ronald Olivenstein Ian D Pavord David McCormack Michel Laviolette Narinder S Shargill Gerard Cox 《BMC pulmonary medicine》2011,11(1):1-9