Biomedical subjects

Z Aguilar

Publications and source records attributed to Z Aguilar.

4 recordsLinked to original sources

PubMed2001

The transmembrane heregulin precursor is functionally active.

A variety of eucaryotic polypeptide growth factors are synthesized as transmembrane precursors. Many of these precursors are released from plasma membranes by proteolytic cleavage and converted into soluble mature proteins. A number of studies, however, indicate that bound growth factor precursors can be biologically active, suggesting a role for these membrane-associated ligands in cell-cell communication. Secreted heregulin is a 45-kDa growth factor with homology to epidermal growth factor. This growth factor binds directly to HER-3 and HER-4 and activates heterodimeric receptor complexes composed of the type I receptor tyrosine kinases, i.e. HER-1, HER-2, HER-3, and HER-4. Heregulin was originally detected in the conditioned medium of the human breast cancer cell line MDA-MB-231 and purified based on its ability to stimulate phosphorylation of p185(HER-2/neu). In the current study, the biologic activity of plasma membrane-anchored heregulin was evaluated in human breast cells. Transmembrane heregulin binds to cells expressing p180(HER-3), induces p185(HER-2/neu) phosphorylation, and increases DNA synthesis in cells overexpressing the HER-2/neu gene product. In addition, when cells containing heregulin receptors are co-cultured with heregulin-producing cells, specific in vivo associations are observed. This study demonstrates that transmembrane heregulin is functionally active and suggest it is capable of playing a role in cell-cell communication and subsequent signal transduction in vivo.

Cell Membrane↗

PubMed1999

Biologic effects of heregulin/neu differentiation factor on normal and malignant human breast and ovarian epithelial cells.

The heregulins are a family of ligands with ability to induce phosphorylation of the p185HER-2/neu receptor. Various investigators have reported a variety of responses of mouse and human breast and ovarian cells to this family of ligands including growth stimulation, growth inhibition, apoptosis and induction of differentiation in cells expressing the HER-2/neu receptor. Some of the disparity in the literature has been attributed to variations in the cell lines studied, ligand dose applied, methodologies utilized or model system evaluated (i.e. in vitro or in vivo). To evaluate the effects of heregulin on normal and malignant human breast and ovarian epithelial cells expressing known levels of the HER-2/neu receptor, this report presents the use of several different assays, performed both in vitro and in vivo, in vitro proliferation assays, direct cell counts, clonogenicity under anchorage-dependent and anchorage-independent conditions, as well as the in vivo effects of heregulin on human cells growing in nude mice to address heregulin activity. Using a total of five different biologic assays in nine different cell lines, across two different epithelia and over a one log heregulin dose range, we obtained results that clearly indicate a growth-stimulatory role for this ligand in human breast and ovarian epithelial cells. We find no evidence that heregulin has any growth-inhibitory effects in human epithelial cells. We also quantitated the amount of each member of the type I receptor tyrosine kinase family (RTK I, i.e. HER-1, HER-2, HER-3 and HER-4) in the cell lines employed and correlated this to their respective heregulin responses. These data demonstrate that HER-2/neu overexpression itself affects the expression of other RTK I members and that cells expressing the highest levels of HER-2/neu have the greatest response to HRG.

Animals↗

PubMed1988

Alteration of lipid order profile and permeability of plasma membranes from Trypanosoma cruzi epimastigotes grown in the presence of ketoconazole.

Highly purified preparations of plasma membranes from control and ketoconazole-treated (1 microM, 120 h) epimastigotes of Trypanosoma cruzi have been obtained by cell disruption using abrasion with glass beads, differential centrifugation and isopycnic centrifugation in continuous, self-generating Percoll gradients. The purity of the preparation was ascertained by the specific activity 125I bound to the membranes obtained from enzymatically radiolabeled epimastigotes and by the alpha-methyl-mannoside sensitive binding of 125I-concanavalin A. The membranes form closed vesicles of 0.2-0.4 micron in diameter which display Mg2+ ATPase and acid phosphatase activities, but are devoid of 5'-nucleotidase and succinate-cytochrome c oxidoreductase; these vesicles can be strongly agglutinated by concanavalin A. The lipid order profiles of membranes from control and treated cells were compared with that present in egg phosphatidylcholine/ergosterol liposomes (84:16, mol/mol) by electron spin resonance spectroscopy of doxylstearic acid probes with the nitroxide group bound to carbon 5, 10, 12 and 16 of the stearic acid chain. Membranes from treated epimastigotes have a lipid order profile which resembles that of control plasma membranes near the polar surface (positions 5 and 10) but there is an abrupt decrease of order at position 12 and from there to the center of bilayer is highly disordered, even more than in pure lipid membranes. Consistent with these results, the leakage of L-[14C]glucose from membrane vesicles of ketoconazole-treated cells is much faster than that observed in vesicles obtained from control cells. These results indicate a strong alteration of the plasma membrane physical and biological properties due to the incubation of the parasite with the drug; this alteration is consistent with the accumulation of methylated precursors of ergosterol, which affects both lipid-lipid and lipid-protein interactions in the membrane.

Animals↗

PubMed1986

The phosphofructokinase of Trypanosoma (Schizotrypanum) cruzi: purification and kinetic mechanism.

The phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, E.C.2.7.1.11) of Trypanosoma (Schizotrypanum) cruzi epimastigotes has been purified 180-fold, to apparent electrophoretic homogeneity, by differential centrifugation, gel filtration chromatography and anionic exchange chromatography. The minimum catalytic unit of the purified enzyme is a polypeptide of 17,000 +/- 1300 molecular weight, as shown by gel filtration chromatography and SDS-gel electrophoresis. Hanes-Woolf plots of initial rates and Mg-ATP or D-fructose-6-phosphate concentrations for varying values of the co-substrate concentration gave intersecting lines which indicate a sequential mechanism. No inhibition is observed at high Mg-ATP concentrations, confirming the result of a previous study using a partially purified enzyme. The pure enzyme displays both negative and positive cooperative kinetics at low (less than 0.8 mM) concentrations of D-fructose-6-phosphate, suggesting aggregation and/or activation phenomena induced by this substrate. Product inhibition studies at saturating and non-saturating concentrations of D-fructose-6-phosphate gave as the only compatible mechanism an iso-ordered bi-bi process with a probable order of entry to and exit from the active site being: D-fructose-6-phosphate followed by Mg-ATP and D-fructose-1,6-bisphosphate followed by Mg-ADP, respectively. The very important differences in both the structural and mechanistic aspects between this enzyme and its vertebrate and bacterial counterparts supports the notion of a highly unusual carbohydrate catabolism in this parasite.

Animals↗