Browsing by Author "González, A"

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    A dichotomous species of Codium (Bryopsidales, Chlorophyta) is colonizing northern Chile
    (2004) González, A; Santelices, B
    In late 2001 and early 2002, a dichotomous species of Codium appeared colonizing the low intertidal and shallow subtidal bottoms of Caldera Bay, northern Chile (27degrees03' S, 70degrees51' W). Due to the ecological and economic impact the species is having in Caldera Bay and its potential spread along the Chilean coastline, we studied the taxonomic identity of the species and examined its relationships with other dichotomous species of Codium reported for temperate Pacific South America. Morphological analyses suggest that the seaweeds from Caldera Bay belong to Codium fragile (Suringar) Hariot. Not only is there strong agreement in internal and external morphological characters, but among all the species reported for Peru and Chile, this is the only one exhibiting utricles with rounded, apiculate tip terminating in a mucron. This species has a broad geographic distribution in temperate waters. In Chile it was known only from the coasts of Valdivia to the Straits of Magellan (39degrees48' S, 73degrees26' W to 53degrees10' S, 73degrees49' W). This is the first record of C. fragile in northern Chile, and this study discusses several alternative hypotheses for the presence of the species into this area. The morphological characteristics of the material collected in Caldera partially agree with diagnostic characters known for C. fragile subspecies tasmanicum and C. fragile subspecies tomentosoides. However, the rapid population spread of the species in northern Chile, and recent molecular analysis support the identification of this form as the invasive C. fragile subspecies tomentosoides.
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    Antiribosomal P protein antibodies in Chilean SLE patients
    (2002) Massardo, L; Burgos, P; Martínez, ME; Pérez, R; Calvo, M; Barros, M; González, A; Jacobelli, S
    The objective of this work was to determine the frequency and clinical associations of antiribosomal P protein antibodies (Anti-P) in a cohort of Chilean patients with systemic lupus erythematosus (SLE). Between 1996 and 1998, 141 consecutive patients with SLE,here examined prospectively according with a standard protocol. Disease activity was measured by MEX-SLEDAI in 138 patients. Anti-P positivity was determined by double immune diffusion or Western blot and ELISA. Anti-P was found in 21 (15%) patients, In the Anti-P positive patients recent onset SLE (disease duration of 1 year or less) was more frequent (P = 0.018). Anti-P was found in 73% of 83 patients with active SLE vs 4% of the 55 patients with inactive SLE (Yates corrected P = 0.00479). An association with anti-dsDNA antibodies by Farr assay was observed. Anti-P positive patients had a median Farr of 65IU/ml (1.4-1240) and Anti-P negative of 12IU/ml (1.4-992 P-value = 0.0084). During the study only two patients had lupus psychosis and they were Anti-P positive. No association,vas found with liver disease (six patients, two with Anti-P antibodies) or active glomerulonephritis (22 patients, four with Anti-P). Our data shows that the presence of Anti-P antibodies supports the clinical diagnosis of lupus psychosis.
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    Apical sorting of a voltage- and Ca2+-activated K+ channel α-subunit in Madin-Darby canine kidney cells is independent of N-glycosylation
    (2000) Bravo-Zehnder, M; Orio, P; Norambuena, A; Wallner, M; Meera, P; Toro, L; Latorre, R; González, A
    The voltage- and Ca2+-acttiated K+ (K-V,K-Ca) channel is expressed in a variety of polarized epithelial cells seemingly displaying a tissue-dependent apical-to-basolateral regionalization, as revealed by electrophysiology. Using domain-specific biotinylation and immunofluorescence we show that the human channel K-V,K-Ca alpha -subunit (human Slowpoke channel, hSlo) is predominantly found in the apical plasma membrane domain of permanently transfected Madin-Darby canine kidney cells. Both the wild-type and a mutant hSlo protein lacking its only potential N-glycosylation site were efficiently transported to the cell surface and concentrated in the apical domain even when they were overexpressed to levels 200- to 300-fold higher than the density of intrinsic Slo channels. Furthermore, tunicamycin treatment did not prevent apical segregation of hSlo, indicating that endogenous glycosylated proteins (e.g., K-V,K-Ca beta -subunits) were not required. hSlo seems to display properties for lipid-raft targeting, as judged by its buoyant distribution in sucrose gradients after extraction with either detergent or sodium carbonate. The evidence indicates that the hSlo protein possesses intrinsic information for transport to the apical cell surface through a mechanism that may involve association with lipid rafts and that is independent of glycosylation of the channel itself or an associated protein. Thus, this particular polytopic model protein shows that glycosylation-independent apical pathways exist for endogenous membrane proteins in Madin-Darby canine kidney cells.
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    EGF receptor transactivation by urokinase receptor stimulus through a mechanism involving Src and matrix metalloproteinases
    (2004) Guerrero, J; Santibañez, JF; González, A; Martínez, J
    Urokinase-type plasminogen activator receptor (uPAR) and epidermal growth factor receptor (EGFR) are ubiquitous receptors involved in the control of a variety of cellular processes frequently found altered in cancer cells. The EGFR has been recently described to play a transduction role of uPAR stimuli, mediating uPA-induced proliferation in highly malignant cells that overexpress uPAR. In the present work, we found for the first time that uPAR stimulation with the amino-terminal fragment (ATF) of urokinase devoid of proteolytic activity transactivates the EGFR in mammary MCF-7 cells through a mechanism involving Src and a metalloproteinase, as indicated by its sensitivity to selected inhibitors. In these cells, which express low levels of uPAR and malignancy, both ATF and EGF stimuli induced an interaction of the EGFR with uPAR and ERK activation. However, EGFR activation by uPAR stimuli mediated cellular invasion rather than proliferation, while EGFR activation by EGF led to a proliferative response. These results revealed a complex modulation of EGFR function toward different cellular responses according to the status of uPAR activity. On the other hand, we also found that NIMP-mediated activation of EGFR can occur in an autocrine manner in cells which secrete uPA. All this reveals novel regulatory systems operating through autocrine loops involving uPAR stimuli, Src, MMP and EGFR activation which could mediate fine control of physiological processes as well as contribute to the expression of proliferative and invasive phenotypes of cancerous cells. (C) 2003 Elsevier Inc. All rights reserved.
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    Increased expression of c-rel, from the NF-κB/Rel family, in T cells from patients with systemic lupus erythematosus
    (2000) Burgos, P; Metz, C; Bull, P; Pincheira, R; Massardo, L; Errázuriz, C; Bono, MR; Jacobelli, S; González, A
    Objective. To explore the role of the NF-kappa B/Rel transcription factor family in autoimmunity, we investigated whether peripheral blood mononuclear cells (PBMC) and T cells from the blood of patients with systemic lupus erythematosus (SLE) exhibit abnormal expression of c-rel, both when recently isolated and/or during in vitro activation.
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    Novel mechanism for regulation of epidermal growth factor receptor endocytosis revealed by protein kinase A inhibition
    (2002) Salazar, G; González, A
    Current models put forward that the epidermal growth factor receptor (EGFR) is efficiently internalized via clathrin-coated pits only in response to ligand-induced activation of its intrinsic tyrosine kinase and is subsequently directed into a lysosomal-proteasomal degradation pathway by mechanisms that include receptor tyrosine phosphorylation and ubiquitylation. Herein, we report a novel mechanism of EGFR internalization that does not require ligand binding, receptor kinase activity, or ubiquitylation and does not direct the receptor into a degradative pathway. inhibition of basal protein kinase A (PKA) activity by H89 and the cell-permeable substrate peptide Myr-PKI induced internalization of 40-60% unoccupied, inactive EGFR, and its accumulation into early endosomes without affecting endocytosis of transferrin and A-opioid receptors. This effect was abrogated by interfering with clathrin function. Thus, the predominant distribution of inactive EGFR at the plasma membrane is not simply by default but involves a PKA-dependent restrictive condition resulting in receptor avoidance of endocytosis until it is stimulated by ligand. Furthermore, PKA inhibition may contribute to ligand-induced EGFR endocytosis because epidermal growth factor inhibited 26% of PKA basal activity. On the other hand, H89 did not alter ligand-induced internalization of EGFR but doubled its half-time of clown-regulation by retarding its segregation into degradative compartments, seemingly due to a delay in the receptor tyrosine phosphorylation and ubiquitylation. Our results reveal that PKA basal activity controls EGFR function at two levels: 1) residence time of inactive EGFR at the cell surface by a process of "endocytic evasion," modulating the accessibility of receptors to stimuli; and 2) sorting events leading to the down-regulation pathway of ligand-activated EGFR, determining the length of its intracellular signaling. They add a new dimension to the fine-tuning of EGFR function in response to cellular demands and cross talk with other signaling receptors.
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    Relationship Between Nostril, Nasal Valve and Minimal Cross-Sectional Area in Functional Upper Airway
    (2019) Farina, R.; González, A; Toledo, X.; Villanueva Conejeros, Rodrigo Ricardo; Martínez, B.; Pérez, H.
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    Selective plasma membrane permeabilization by freeze-thawing and immunofluorescence epitope access to determine the topology of intracellular membrane proteins
    (2003) Mardones, G; González, A
    The structural and functional characterization of membrane proteins includes assessment of their topology in the bilayer. In the present work, we successfully used an approach based on comparative epitope accessibility. The classical method of detergent permeabilization of fixed cells allowed antibodies to detect epitopes distributed at either side of each cellular membrane by immunofluorescent staining. Instead, freeze-thawing followed by fixation allowed antibodies to cross only the plasma membrane whereas all intracellular membranes remained impermeable. By combining the immunofluorescence results achieved with these two methods for a variety of known membrane proteins, we showed that epitope accessibility could be accurately determined in proteins residing in the plasma membrane or in intracellular compartments, including the endoplasmic reticulum, lysosomes, peroxisomes, different Golgi regions and the nucleus. Freeze-thawing neither changed the expected distribution of each tested protein nor permeabilized intracellular membranes to antibodies. It only permeabilized the plasma membrane. Furthermore, the protocol proved to be efficient in different kinds of cells, which include MDCK and FRT polarized epithelial cells, HeLa cells and fibroblasts. If the complete topology of an integral membrane protein is known, this method would allow to assign an orientation to epitopes recognized by a panel of monoclonal antibodies. It also avoids the use of toxic reagents for permeabilization. Thus, selective permeabilization of the plasma membrane by freeze-thawing provides an inexpensive and reliable method to investigate the topology of membrane proteins as well as the distribution of soluble proteins. (C) 2003 Elsevier Science B.V. All rights reserved.
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    Sorting competition with membrane-permeable peptides in intact epithelial cells revealed discrimination of transmembrane proteins not only at the trans-Golgi network but also at pre-Golgi stages
    (2004) Soza, A; Norambuena, A; Cancino, J; de la Fuente, E; Henklein, P; González, A
    Transmembrane proteins destined to the basolateral cell surface of epithelial cells contain in their cytosolic domain at least two classes of sorting signals: one class promotes exit from the endoplasmic reticulum (ER) and transport to the Golgi complex, and the other class operates at the trans-Golgi network (TGN) specifying segregation into basolateral exocytic pathways. Both kinds of addressing motifs are quite diverse among different proteins. It is unclear to what extent this feature reflects alternative decoding mechanisms or variations in motifs recognized by the same sorting factor. Here we applied a novel strategy based on permeable peptide technology and temperature-sensitive model proteins to study competition between cytosolic sorting motifs in the context of mammalian living cells. We used the transduction domain of HIV-1 Tat protein to make a membrane-permeable peptide of the cytosolic tail of GtsO45, which contains a well characterized ER exit di-acidic (DIE) motif and a tyrosine-based basolateral sorting signal (YTDI). This peptide added to the media inhibited transport of GtsO45 from both ER-to-Golgi and TGN-to-basolateral cell surface in transfected Madin-Darby canine kidney cells. Instead, it did not affect the exocytic trafficking of a GtsO45-derived chimeric protein bearing 30 juxtamembrane residues from the cytosolic domain of the epidermal growth factor receptor that contains a variant ER exit motif (ERE) and an unconventional proline-based basolateral sorting signal. These results not only proved the feasibility of competing for sorting events in intact cells but also showed that distinct plasma membrane proteins can be discriminated at pre-TGN stages, and that basolateral sorting involves different recognition elements for tyrosine-based motifs and an unconventional basolateral motif.
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    Two homozygous mutations in the 11β-hydroxysteroid dehydrogenase type 2 gene in a case of apparent mineralocorticoid excess
    (2003) Carvajal, CA; Gonzalez, AA; Romero, DG; González, A; Mosso, LM; Lagos, ET; Hevia, MD; Rosati, MP; Perez-Acle, TO; Gomez-Sanchez, CE; Montero, JA; Fardella, CE
    The human microsomal 11beta-hydroxysteroid dehydrogenase type 2 (11betaHSD2) metabolizes active cortisol into cortisone and protects the mineralocorticoid receptor from glucocorticoid occupancy. In a congenital deficiency of 11beta-HSD2, the protective mechanism fails and cortisol gains inappropriate access to mineralocorticoid receptor, resulting in low-renin hypertension and hypokalemia. In the present study, we describe the clinical and molecular genetic characterization of a patient with a new mutation in the HSD11B2 gene. This is a 4-yr-old male with arterial hypertension. The plasma renin activity and serum aldosterone were undetectable in the presence of a high cortisol to cortisone ratio. PCR amplification and sequence analysis of HSD11B2 gene showed the homozygous mutation in exon 4 Asp223Asn (GAC --> AAC) and a single nucleotide substitution C-->T in intron 3. Using site-directed mutagenesis, we generated a mutant 11betaHSD2 cDNA containing the Asp223Asn mutation. Wild-type and mutant cDNA was transfected into Chinese hamster ovary cells and enzymatic activities were measured using radiolabeled cortisol and thin-layer chromatography. The mRNA and 11betaHSD2 protein were detected by RT-PCR and Western blot, respectively. Wild-type and mutant 11betaHSD2 protein was expressed in Chinese hamster ovary cells, but the mutant enzyme had only 6% of wildtype activity. In silico 3D modeling showed that Asp223Asn changed the enzyme's surface electrostatic potential affecting the cofactor and substrate enzyme-binding capacity. The single substitution C-->T in intron 3 (IVS3 + 14 C-->T) have been previously reported that alters the normal splicing of pre-mRNA, given a nonfunctional protein. These findings may determine the full inactivation of this enzyme, explaining the biochemical profile and the early onset of hypertension seen in this patient.