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61.
Deise Juliana Kolling Jean Borges Bertoldo Fábio Cristiano Angonesi Brod Javier Vernal Hernán Terenzi Ana Carolina Maisonnave Arisi 《Molecular biotechnology》2010,46(2):168-175
Staphylococcus xylosus AF208229 lipase was expressed in E. coli containing an histidine-tag (WT-Val). In the present work, in order to check the importance of the residue 309 in the specific
activity, the amino acid side chain residue valine 309 was substituted by aspartate or lysine through site-directed mutagenesis.
Both mutant lipases (MUT-Lys and MUT-Asp) were expressed in E. coli and the recombinant histidine-tagged lipases were purified by immobilized metal ion affinity chromatography. The enzyme activity
was determined using p-nitrophenyl butyrate as substrate and secondary structure content was evaluated by circular dichroism. MUT-Lys and MUT-Asp
presented significant increase of lipase activity (P < 0.05) in comparison to WT-Val, although highest activities for the three enzymes were observed at the same pH and temperature
(pH 9.0 and 42°C). The wild type and mutant lipases presented high thermal stability, after 30 min of incubation at 80°C all
enzymes retained their initial activities. 相似文献
62.
Alloatti G Marcantoni A Levi R Gallo MP Del Sorbo L Patrucco E Barberis L Malan D Azzolino O Wymann M Hirsch E Montrucchio G 《FEBS letters》2005,579(1):133-140
Cardiac beta-adrenergic and the muscarinic receptors control contractility and heart rate by triggering multiple signaling events involving downstream targets like the phosphoinositide 3-kinase gamma (PI3Kgamma). We thus investigated whether the lack of PI3Kgamma could play a role in the autonomic regulation of the mouse heart. Contractility and ICaL of mutant cardiac preparations appeared increased in basal conditions and after beta-adrenergic stimulation. However, basal and beta-adrenergic stimulated heart rate were normal. Conversely, muscarinic inhibition of heart rate was reduced without alteration of the Gbetagamma-dependent stimulation of IK,ACh current. In addition, muscarinic-mediated anti-adrenergic effect on papillary muscle contractility and ICaL was significantly depressed. Consistently, cAMP level of PI3Kgamma-null ventricles was always higher than wild-type controls. Thus, PI3Kgamma controls the cardiac function by reducing cAMP concentration independently of Gi-mediated signaling. 相似文献
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64.
Neospora caninum excreted/secreted antigens trigger CC-chemokine receptor 5-dependent cell migration
Tiago W.P. Mineo Carlo J.F. Oliveira Deise A.O. Silva Antônio R. Abatepaulo Beatriz R. Ferreira João S. Silva 《International journal for parasitology》2010,40(7):797-805
Neospora caninum, the causative agent of neosporosis, is an obligate intracellular parasite considered to be a major cause of abortion in cattle throughout the world. Most studies concerning N. caninum have focused on life cycle, seroepidemiology, pathology and vaccination, while data on host-parasite interaction, such as host cell migration, mechanisms of evasion and dissemination of this parasite during the early phase of infection are still poorly understood. Here we show the ability of excreted/secreted antigens from N. caninum (NcESAs) to attract monocytic cells to the site of primary infection in both in vitro and in vivo assays. Molecules from the family of cyclophilins present on the NcESAs were shown to work as chemokine-like proteins and NcESA-induced chemoattraction involved Gi protein signaling and participation of CC-chemokine receptor 5 (CCR5). Additionally, we demonstrate the ability of NcESAs to enhance the expression of CCR5 on monocytic cells and this increase occurred in parallel with the chemotactic activity of NcESAs by increasing cell migration. These results suggest that during the first days of infection, N. caninum produces molecules capable of inducing monocytic cell migration to the sites of infection, which will consequently enhance initial parasite invasion and proliferation. Altogether, these results help to clarify some key features involved in the process of cell migration and may reveal virulence factors and therapeutic targets to control neosporosis. 相似文献