RESUMO
Trypanosoma cruzi, the etiologic Chagas' disease agent, induces changes in protein pattern of the human placenta syncytiotrophoblast. The glucose transporter protein-1 (GLUT1) is the primary isoform involved in transplacental glucose transport. We carried out in vitro assays to determine if T. cruzi infection would induce changes in placental GLUT1 protein expression under normal and high concentration of glucose. Using Western blot and immunohistological techniques, GLUT1 expression was determined in normal placental villi cultured under normal or high concentrations of glucose, with or without in vitro T. cruzi infection, for 24 and 48 hours. High glucose media or T. cruzi infection alone reduced GLUT1 expression. A yet more accentuated reduction was observed when infection and high glucose condition took place together. We inform, for the first time, that T. cruzi infection may induce reduction of GLUT1 expression under normal and high glucose concentrations, and this effect is synergic to high glucose concentrations.
RESUMO
Alkaline phosphatases (APs) catalyze the hydrolysis and transphosphorylation of phosphate monoesters. Quantum mechanical, molecular dynamics, and molecular docking techniques were applied to computationally model the catalytic mechanism of human placental AP (PLAP). Kinetic and thermodynamic evaluations were performed for each reaction step. The functional significances of the more important residues within the active site were analyzed. The role of the metal ion at the metal binding site M3 was also examined. The calculated activation and reaction energy and free energy values obtained suggested the nucleophilic attack of the Ser92 alkoxide on the phosphorus atom of the substrate would be the rate-limiting step of the catalytic hydrolysis of alkyl phosphate monoesters by PLAP. The reactivities of the wild-type M3-Mg enzyme and the M3-Zn protein were compared, and the main difference observed was a change in the coordination number of the M3 metal for the M3-Zn enzyme. This modification in the active site structure lowered the free energy profile for the second chemical step of the catalytic mechanism (hydrolysis of the covalent phosphoserine intermediate). Consequently, a greater stabilization of the phosphoseryl moiety resulted in a small increment in the activation free energy of the phosphoserine hydrolysis reaction. These computational results suggest that the activation of APs by magnesium at the M3 site is caused by the preference of Mg(2+) for octahedral coordination, which structurally stabilizes the active site into a catalytically most active conformation. The present theoretical results are in good agreement with previously reported experimental studies.
Assuntos
Fosfatase Alcalina/química , Fosfatase Alcalina/metabolismo , Biocatálise , Simulação por Computador , Isoenzimas/química , Isoenzimas/metabolismo , Domínio Catalítico , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/metabolismo , Humanos , Cinética , Magnésio/metabolismo , Simulação de Dinâmica Molecular , Teoria Quântica , Termodinâmica , Zinco/metabolismoRESUMO
Trypanosoma cruzi induces changes in the protein pattern of human placenta syncytiotrophoblast. Placental alkaline phosphatase (PLAP) is a glycoenzyme anchored to the membrane by a glycosyl-phosphatidylinositol molecule. PLAP activity and its presence was altered by the parasite in cultures of human placental villi and HEp2 cells with T.cruzi. The cells treated before the cultures with agents which affect PILAP or glycosyl-phosphatidylinositol (antibodies, PL-C, genistein, lithium) presented less parasitic invasion than the control ones. It was also observed a modification in the pattern of actine filaments of the host cells infected. We concluded that PLAP would participate in the process of T. cruzi invasion into placental syncitiotrophoblast cells, by a mechanism that involves hydrolysis of the glycosyl-phosphatidylinositol molecules, the activation of tyrosine kinase proteins, the increase of cytosolic calcium and the rearrangement of actine filaments of the host cells.
Assuntos
Fosfatase Alcalina/metabolismo , Doença de Chagas/enzimologia , Placenta/enzimologia , Trypanosoma cruzi/fisiologia , Fosfatase Alcalina/análise , Análise de Variância , Animais , Biomarcadores , Técnicas de Cultura de Células , Doença de Chagas/imunologia , Doença de Chagas/parasitologia , Vilosidades Coriônicas/enzimologia , Vilosidades Coriônicas/parasitologia , Feminino , Glicosilfosfatidilinositóis/metabolismo , Humanos , Imuno-Histoquímica , Placenta/parasitologia , Gravidez , Trofoblastos/enzimologia , Trofoblastos/parasitologiaRESUMO
The Kinetic properties of plasma placental alkaline phosphatase patients with Chagas' disease were studied. When Cl2 Mg was used as activator the same increase of activity (17-20 per cent) was found in the chagasic and non chagasic groups. The enzyme was not inhibited by F-ion in any of the groups. No significant differences were detected between the two groups (chagasic and non chagasic) when the enzyme was treated with inhibitors such as EDTA and L-phenylamine. However, when the CN- ion was used, the enzyme of the normal pregnant women followed a Michaelian curve, whereas in the chagasic group a sigmoideal plot was observed. Thus, the Hill coefficient was 1.1 for the normal group and over 1.5 for the chagasic.
Assuntos
Adulto , Feminino , Humanos , Gravidez , Fosfatase Alcalina/sangue , Doença de Chagas/enzimologia , Ácido Edético , Placenta/enzimologia , Complicações Parasitárias na Gravidez/enzimologia , Fosfatase Alcalina/efeitos dos fármacos , Fosfatase Alcalina/metabolismo , Doença de Chagas/sangue , Ácido Edético , Inibidores Enzimáticos/farmacologia , Reativadores Enzimáticos/farmacologia , Complicações Parasitárias na Gravidez/sangue , Terceiro Trimestre da GravidezRESUMO
The Kinetic properties of plasma placental alkaline phosphatase patients with Chagas disease were studied. When Cl2 Mg was used as activator the same increase of activity (17-20 per cent) was found in the chagasic and non chagasic groups. The enzyme was not inhibited by F-ion in any of the groups. No significant differences were detected between the two groups (chagasic and non chagasic) when the enzyme was treated with inhibitors such as EDTA and L-phenylamine. However, when the CN- ion was used, the enzyme of the normal pregnant women followed a Michaelian curve, whereas in the chagasic group a sigmoideal plot was observed. Thus, the Hill coefficient was 1.1 for the normal group and over 1.5 for the chagasic. (AU)