Browsing by Author "GONZALEZ, A"

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    APICAL SECRETION OF HEPATITIS-B SURFACE-ANTIGEN FROM TRANSFECTED MADIN-DARBY CANINE KIDNEY-CELLS
    (AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 1993) GONZALEZ, A; NICOVANI, S; JUICA, F
    Hepatitis B surface antigen (HBsAg), the major envelope component of human hepatitis B virus, during infection drives the assembly and basolateral secretion from hepatocytes of both virions and subviral lipoprotein particles into the bloodstream. We studied the sorting behavior of HBsAg in the heterologous epithelial Madin-Darby canine kidney cells permanently transformed with the hepatitis B virus S gene. These cells, forming tightly packed monolayers in permeable supports, secreted HBsAg apically through a mechanism not involving transcytosis. This suggests that molecular features acting as apical addressing information, seemingly unfunctional or less efficiently used by the exocytic machinery of hepatocytes, could be contained in short hydrophilic regions of HBsAg. Lipids also could play a role in this asymmetric sorting because HBsAg is known to be secreted by forming macromolecular lipoprotein complexes rather than as a soluble protein. Together with available data, our results would imply not only the existence of tissue-specific variations in handling constitutively secreted proteins but also that these variations are strikingly dependent on the kind of protein examined. On the other hand, pulse-chase experiments with tunicamycin showed that the expression of apical information in HBsAg particles does not require N-linked glycosylation, contrasting with the known gp80 Madin-Darby canine kidney-endogenous apical secretory marker. This is the first experimental evidence that carbohydrate moieties in secretory proteins do not hold domain-specific sorting signals, a fact previously shown exclusively for membrane proteins. Thus, HBsAg provides a novel model system for the analysis of the molecular mechanisms of constitutive apical secretion.
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    CELL-GROWTH AND NA-K-CL COTRANSPORT RESPONSES OF VASCULAR SMOOTH-MUSCLE CELLS OF MILAN RATS
    (LIPPINCOTT WILLIAMS & WILKINS, 1994) CANESSA, M; SALAZAR, G; WERNER, E; VALLEGA, G; GONZALEZ, A
    The present study examines the role of serum growth factors in the proliferative response and Na-K-Cl cotransport activity of vascular smooth muscle cells from Milan normotensive (MNS) and hypertensive (MHS) rats. Cells from thoracic aorta of both strains were cultured in 10% serum medium and made quiescent by 72 hours in 0.3% serum medium. MHS cells grown with 10% serum had a shorter population doubling time than MNS cells between passages 8 and 12 (13.8+/-1.7 versus 20.1+/-1.6 hours, P<.0l, n=4). MHS cells also exhibited a higher response of thymidine incorporation into nucleic acid to serum, epidermal, and platelet-derived growth factor BB. In MHS cells epidermal (100 ng/mL) and platelet (50 ng/mL) growth factors increased thymidine incorporation 2- and 10-fold, respectively. In MNS cells epidermal factor did not induce a significant response, and that of platelet factor was twofold lower than in MHS cells. Binding curves revealed a higher number of receptors for platelet than epidermal growth factor in both strains and a similar number of both receptors in MHS and MNS cells. Quantitative immunoblots of these receptor proteins confirmed the observation that the greater proliferation of MHS cells could not be related to a higher number of growth factor receptors. Cotransport activity (bumetanide-sensitive Rb-86 influx in nanomoles per milligram protein per 5 minutes) was found to be significantly higher in MHS cells (16+/-3, n=18) than MNS cells (8+/-3, n=15) at confluence as well as in the log phase of serum-stimulated growth. No differences were found between strains with cells in the quiescent state. However, platelet growth factor (50 ng/mL) markedly stimulated cotransport in quiescent MHS cells, whereas epidermal growth factor was without effect. In conclusion, MHS cells exhibited enhanced serum- and platelet factor-stimulated proliferation rates and cotransport activity compared with MNS cells. These results suggest that platelet factor BB plays an important role in the proliferation of hypertensive vascular cells.
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    CIPROFIBRATE, A CARCINOGENIC PEROXISOME PROLIFERATOR, INCREASES THE PHOSPHORYLATION OF EPIDERMAL-GROWTH-FACTOR RECEPTOR IN ISOLATED RAT HEPATOCYTES
    (1993) ORELLANA, A; HOLUIGUE, L; HIDALGO, PC; FAUNDEZ, V; GONZALEZ, A; BRONFMAN, M
    Ciprofibrate, a hypolipidaemic drug with carcinogenic and peroxisome-proliferation effects in rat liver, was found to increase the phosphorylation of epidermal-growth-factor receptor in P-32-labeled isolated rat hepatocytes. This effect was suppressed by protein-kinase-C inhibitors, and was accompanied by an almost complete inhibition of the receptor autophosphorylation normally induced by its ligand. However, in vitro experiments showed that protein-kinase-C phosphorylation of purified epidermal-growth-factor receptor was activated by ciprofibroyl-CoA, the acyl-CoA derivative of the drug, but not by the unmodified drug. Neither compound affected the ligand induction of epidermal-growth-factor-receptor autophosphorylation in isolated liver membranes. These results suggest that metabolically produced ciprofibroyl-CoA in liver cells would activate protein-kinase-C and produce changes in epidermal-growth-factor-receptor function.
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    CLINICAL EXPRESSION OF RHEUMATOID-ARTHRITIS IN CHILEAN PATIENTS
    (W B SAUNDERS CO, 1995) MASSARDO, L; AGUIRRE, V; GARCIA, ME; CERVILLA, V; NICOVANI, S; GONZALEZ, A; RIVERO, S; JACOBELLI, S
    In populations such as Northern Europeans in which the HLA-DR4 subtypes Dw14 and Dw4 show strong association with rheumatoid arthritis (RA), these alleles and the double allelic dose of the shared epitope are considered severity markers. The clinical expression of RA varies in different populations, which may be determined by variation in the prevalence of these markers. In the present study we analyzed the expression of RA in 112 consecutive Chilean patients and its relation to the prevalence of genetic factors, prompted by our previous observation that DR4 is weakly associated to RA in this population. Mean age was 50 +/- 14 years; 90% were seropositive and 87% were female, with a disease duration of 10 +/- 8 years. Extra-articular manifestations were found in 38% of patients, rheumatoid nodules in 27%, vasculitis in 8%, and Sjogren's syndrome in 29%. Functional capacity (ACR, 1991) I or II: 82%. 15% of patients stopped working. Hand radiographs scored according to Steinbrocker in 89 patients: I, 21%; II, 15%; III, 43%; IV, 21%. In this series, patients with less formal education seemed to have more benign arthritis. In 97 controls and in 65 (56%) RA patients the presence of DRB1 alleles corresponding to DR1 and DR4 serotypes, to DR4-Dw subtypes, and homozygocity, were determined by polymerase chain reaction followed by specific oligonucleotide hybridization. The shared epitope was present in 53% of RA patients and in 30% of controls (P = .0048, odds ratio [OR] = 2.64). A double allelic dose of the epitope was present in 15% of RA patients compared with 4% of controls (P = .026, OR = 4.23). In a subgroup of 31 erosive RA patients we did not find a significant association of disease severity with the shared epitope in a single or double allelic dose. None of the DR4 subtypes that associate with RA in other populations was found significantly more prevalent in our patients. The severity of RA in our study compared with published series was intermediate between British patients with severe RA and Creek patients with milder disease. This may be due to the high prevalence of Dw13*0403 in our population.
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    DOES CONTINUOUS POSITIVE AIRWAY PRESSURE (CPAP) DURING WEANING FROM INTERMITTENT MANDATORY VENTILATION IN VERY-LOW-BIRTH-WEIGHT INFANTS HAVE RISKS OR BENEFITS - A CONTROLLED TRIAL
    (1995) TAPIA, JL; BANCALARI, A; GONZALEZ, A; MERCADO, ME
    Objective: The purpose of this study was to evaluate three ventilator weaning strategies and to evaluate whether the use of continuous positive airway pressure (CPAP) via a nasopharyngeal or endotracheal tube would increase the likelihood of extubation failure in very low birth weight (VLBW) infants. Study design: We studied prospectively 87 preterm infants (mean +/- SD; birth weight: 1078 +/- 188 g; gestational age: 28.8 +/- 2.2 weeks) who were in the process of being weaned from intermittent mandatory ventilation (IMV). Infants were assigned by systematic sampling to one of the following three treatment groups: (1) direct extubation from IMV (D.EXT) (n=30); (2) preextubation endotracheal CPAP (ET-CPAP) for 12-24 hr (n=28); or (3) postextubation nasopharyngeal CPAP (NP-CPAP) for 12-24 hr (n=29). Failure was defined as the need for resumption of mechanical ventilation within 72 hr of extubation due to frequent or severe apnea and/or respiratory failure (pH < 7.25, PaCO2 > 60 mm Hg, and/or requirement for oxygen FiO(2) > 60%). Results: There were no significant differences in failure rates among the three procedures. Failures were 2/30 (7%) in D.EXT; 4/28 (14%) in ET-CPAP; and 7/29 (24%) in the NP-CPAP. There were also no differences in FiO(2), PaO2, and respiratory rates before and after discontinuation of IMV among the three groups. PaCO2 values were slightly higher in the NP-CPAP group 12-24 hr after weaning from IMV. Conclusion: We were unable to demonstrate a clear difference in extubation outcome by use of CPAP administered via an endotracheal or nasopharyngeal tube when compared to direct extubation from low-rate IMV in VLBW infants. (C) 1995 Wiley-Liss, Inc.
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    EPIDERMAL GROWTH-FACTOR INHIBITS CYTOSKELETON-RELATED CHANGES IN THE SURFACE OF PARIETAL-CELLS
    (1981) GONZALEZ, A; GARRIDO, J; VIAL, JD
    The effect of epidermal growth factor (EGF) on gastric acid secretion was correlated with the morphological changes of the apical pole of rat parietal cells studied by transmission electron microscopy. Gastric acid secretion was stimulated by histamine, carbachol, pentagastrin and insulin-induced hypoglycemia and estimated by continuous recording of pH variations of gastric luminal perfusate. EGF inhibits acid secretion in these conditions. The action of the hormone also results in the arrest or reversal of the changes in shape undergone by parietal cells as they go into secretion. In view of the evidence involving cytoskeletal elements in the generation of these structural alterations, the action of EGF on gastric acid secretion may be a consequence of a general effect of this hormone on cytoskeletal function.
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    EPIDERMAL GROWTH-FACTOR RECEPTOR IN SYNAPTIC FRACTIONS OF THE RAT CENTRAL-NERVOUS-SYSTEM
    (1992) FAUNDEZ, V; KRAUSS, R; HOLUIGUE, L; GARRIDO, J; GONZALEZ, A
    Functional relationships between epidermal growth factor (EGF) and neural tissues have of late attracted increasing interest. However, in spite of reported EGF effects on neurons, the expression of the EGF receptor (EGF-R) has not yet been unambiguously demonstrated in these cells. This 170-kDa protein bears an intracellular tyrosine kinase domain in which activity is ligand-dependent. We give definitive evidence here for its presence in neonatal and adult rat neurons showing also, for the first time, its binding and functional tyrosine kinase activities in the synaptic region. Immunohistochemistry using a polyclonal antibody prepared against the receptor purified from rat liver showed positive staining localized exclusively to neurons without regionalization to any particular brain zone. Binding studies made in Percoll-obtained synaptosomes revealed specific high affinity I-125-EGF binding sites (K(d), 1.42 x 10(-10) +/- 0.58 M) accounting for 17% of total binding and a great majority of low affinity (K(d), 2.55 x 10(-9) +/- 0.35 M) binding sites. Higher binding capacity was found in synaptosomal fractions obtained from newborn rats. The identity of the synaptosomal EGF binding activity with the 170-kDa EGF-R protein was demonstrated by cross-linking experiments. Furthermore, EGF-Affi-Prep affinity chromatography adsorbs a 170-kDa protein with EGF-R immunoreactivity from whole homogenates of adult rat brain. Phosphorylation assays made in freeze-thawed or intact synaptosomes showed EGF-induced tyrosine phosphorylation in the range of 170-, 126-150-, 124-, 113-, 98-, and 70-kDa proteins including the EGF-R. Thus, the EGF-R/EGF regulatory system could have a role in synaptic function that remains to be explored.
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    NOVEL GENETIC-MARKERS OF RHEUMATOID-ARTHRITIS IN CHILEAN PATIENTS, BY DR SEROTYPING AND RESTRICTION-FRAGMENT-LENGTH-POLYMORPHISM ANALYSIS
    (1992) GONZALEZ, A; NICOVANI, S; MASSARDO, L; BULL, P; RODRIGUEZ, L; JACOBELLI, S
    Objective. The analysis of genetic markers of rheumatoid arthritis (RA) in a population in which the DR4 serotype is not strongly associated with the disease.
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    THE EARLY CHANGES OF PARIETAL-CELL STRUCTURE IN THE COURSE OF SECRETORY ACTIVITY IN THE RAT
    (1985) VIAL, JD; GARRIDO, J; GONZALEZ, A
    The fine structure of the rat parietal cell was studied, at rest and after stimulation by refeeding or insulin administration. Experiments on fixation procedures showed that whenever the fixative contained sucrose at a concentration higher than 0.2 M, the system of cytoplasmic membranes was clearly tubular in arrangement, whereas the omission of sucrose in the fixative usually resulted in a vesicular structure. High-voltage EM of thick sections prepared by conventional techniques or by impregnation with zinc iodide-osmium (ZIO) revealed that the tubules were grouped into fascicles and these formed a feltwork that was especially thick toward the cell apex. The development of the secretory canaliculus after stimulation appeared to take place by an in situ remodeling of the cytoplasmic domain occupied by the tubular system. Cells examined after short periods of stimulation (5-15 min) showed images of the tubular system and of the canalicular structure which differed both from the nonstimulated and from the fully active (30-45 min of stimulation) cell. These features included the formation of wide cisternae and of pericanalicular cytoplasmic trabeculae or laminae, whose fine structure beared close resemblance to the intracanalicular processes in the same cells. These images can be ordered into a hypothetical sequence which may be a model to explain the transformation of the tubular system and intervening cytoplasmic matrix into secretory canaliculus.
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    WEAK ASSOCIATION BETWEEN HLA-DR4 AND RHEUMATOID-ARTHRITIS IN CHILEAN PATIENTS
    (BRITISH MED JOURNAL PUBL GROUP, 1990) MASSARDO, L; JACOBELLI, S; RODRIGUEZ, L; RIVERO, S; GONZALEZ, A; MARCHETTI, R