Browsing by Author "GARRIDO, J"

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    A TAU FRAGMENT CONTAINING A REPETITIVE SEQUENCE INDUCES BUNDLING OF ACTIN-FILAMENTS
    (1993) MORAGA, DM; NUNEZ, P; GARRIDO, J; MACCIONI, RB
    Much indirect evidence suggests that the interconnections of actin microfilaments with the microtubule system are mediated by microtubule-associated proteins (MAPs). In this study we provide new data to support the interaction of a specific tubulin-binding domain on tau with actin in vitro. In actin polymerization assays, the synthetic peptide VRSKIGSTENLKHQPGGG, corresponding to the first repetitive sequence Of tau protein, increased turbidity at 320 nm in a dose-dependent fashion. A salient feature of the tau peptide-induced assembly process is the formation of a large amount of actin filament bundles, as revealed by electron microscopic analysis. An increase in the tau peptide concentration resulted in a proportional increase in the bundling of actin filaments. It is interesting that a gradual decrease of pH within the range 7.6-4.7 resulted in a higher effect Of tau peptide in promoting bundles of actin filaments. A similar pH-dependent effect was observed for tau protein-induced bundling. An analysis of the mechanisms that operate in the peptide induction of actin filament bundles suggests the involvement of electrostatic forces, because the neutralization of epsilon-aminolysyl residues by selective carbamoylation resulted in a complete loss of the peptide induction of actin bundles. The data suggest that a tau repetitive sequence (also found in MAP-2 and MAP-4) containing a common tubulin binding motif may constitute a functional domain on tau for the dynamics of the interconnections between actin filaments and microtubules.
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    ACTIN-DEFICIENT CARDIOMYOPATHY COEXISTING WITH CELIAC-DISEASE - A CHANCE ASSOCIATION
    (1986) CHUAQUI, B; GARRIDO, J; CASANEGRA, P
    A case of idiopathic congestive cardiomyopathy associated with celiac disease is presented. The ultrastructural and electrophoretic examination of myocardial samples showed a selective loss of actin. Electron microscopy revealed the characteristic alterations of enterocyte microvilli seen in celiac disease. The involvement of the microfilament system both in the myocardial sarcomere and in the enterocyte may indicate a pathogenic relation between these alterations. Ultrastructural examination of skeletal muscle tissue showed a good preservation of the cell contractile apparatus.
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    ACTIN-LIKE FILAMENTS AND MEMBRANE REARRANGEMENT IN OXYNTIC CELLS
    (1976) VIAL, JD; GARRIDO, J
    The secretory pole of vertebrate oxyntic cells possesses 2 distinct membrane systems: the apical plasma membrane which presents numerous infoldings, microvilli and processes and a complex tubulovesicular system located in close proximity to the plasma membrane. These 2 membrane systems are generally believed to be interconvertible in relation to the functional state of the cell. To determine the role that filaments may play in the interconversion process, the secretory pole of rat and toad [Bufo spinulosus] oxyntic cells was examined by EM under conditions designed to demonstrate filamentous structures, i.e., slight cellular swelling and incubation with heavy meromyosin. Filaments 50-80 .ANG. in diameter are present in close association with the plasma membrane to which they are connected by regularly spaced bridges. Heavy meromyosin-treated material reveals decorated filaments in topographically corresponding locations. No filaments are seen in association with membranes of the tubulovesicular system. Association with actin-like filaments may be a step in the translocation of membranes from the tubulovesicular system to the plasma membrane.
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    ACTIN-LIKE FILAMENTS IN SYNAPTOSOMES DETECTED BY HEAVY-MEROMYOSIN BINDING
    (1976) INESTROSA, NC; FERNANDEZ, HL; GARRIDO, J
    Cat forebrain synaptosomes were prepared to investigate the presence of actin-like proteins. Actin-like filaments were demonstrated using a heavy meromyosin binding technique. Decorated filaments form networks inside the synaptosomes and were frequently seen in relation to membranes and synaptic vesicles. Using SDS[sodium dodecylsulfate]-polyacrylamide gel electrophoresis, a band corresponding to actin appeared to be one of the major constituents of synaptosomal fractions; this confirmed the work of Fine and coworkers. The significance of actin-like filaments in relation to axoplasmic transport was briefly discussed.
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    BILIARY LIPID SECRETION - IMMUNOLOCALIZATION AND IDENTIFICATION OF A PROTEIN ASSOCIATED WITH LAMELLAR CHOLESTEROL CARRIERS IN SUPERSATURATED RAT AND HUMAN BILE
    (1993) RIGOTTI, A; NUNEZ, L; AMIGO, L; PUGLIELLI, L; GARRIDO, J; SANTOS, M; GONZALEZ, S; MINGRONE, G; GRECO, A; NERVI, F
    Feeding a 0.5% diosgenin plus 0.02% simvastatin diet to rats increases biliary cholesterol concentration and saturation to levels generally found in human native supersaturated bile. By using preparative ultracentrifugation, gel filtration chromatography, and electron microscopy, we isolated, purified, and identified lamellar structures (unilamellar vesicles and multilamellae) as a major biliary cholesterol transport in supersaturated human and rat bile. It was estimated that more than 60% of biliary cholesterol is transported in these lamellar carriers, which were identified by transmission electron microscopy as unilamellar vesicles and multilamellar bodies within bile canaliculi of rats with cholesterol supersaturated bile. By SDS-PAGE, a characteristic and constant protein profile was found associated to the purified lamellar carriers. One of these proteins, a 130-kDa protein, was isolated from human biliary lamellae and used for preparation of a rabbit polyclonal antibody, which cross-reacted with the homologous rat protein. By Western blotting, it was established that the purified low density fraction of bile-Metrizamide gradients, containing lamellae, was enriched with the 130-kDa protein. The 130-kDa protein was characteristically detected at the canalicular membrane by Western blotting of hepatic subcellular fractions and by immunohistochemistry of rat and human liver biopsies. Amino acid sequencing of the amino terminus of the 130-kDa protein demonstrated a complete identity with aminopeptidase N, a canalicular transmembrane hydrophobic glycoprotein. These studies show that biliary lipids may acquire an ordered multilamellar structure that is present in the canaliculi of rats with supersaturated bile. These biliary lamellae are similar to lamellar bodies and surfactant-like material frequently found in other epithelia, suggesting common biogenetic, structural, and functional properties. The identification of aminopeptidase N associated with biliary lamellae is consistent with the involvement of the canalicular membrane in the secretory mechanism of biliary lipids.
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    CHARACTERIZATION OF PEROXISOMES IN CHINESE-HAMSTER OVARY CELLS IN CULTURE
    (1985) SANTOS, MJ; GARRIDO, J; OLIVER, C; ROBBINS, AR; LEIGHTON, F
    In order to explore the potential value of Chinese hamster overay (CHO) cells for the isolation of peroxisomal mutants defective in the peroxisomal fatty acid oxidation system, some characteristics of their peroxisomes were studied. Catalase was detected biochemically and histochemically in peroxisome-like particles in cells or in subcellular fractions prepared by differential centrifugation or isopyknic equilibrium in Percoll or Metrizamide with catalase in the high density fraction of the isopyknic equilibrium gradients. By oxidation system, exhibited an unusually high specific activity, 2.46 .+-. 1.09 mU/mg protein, in CHO cell homogenates, a value comparable to that of rat liver. This enzyme copurifies with catalase in the high density fractions of the isopycnic equilibrium gradients. By analogy with other cell types and from the ultrastructural analysis, it is concluded that these enzymes are contained in peroxisomes. These findings support the value of CHO cells for studies of peroxisomal function and organization.
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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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    GREEN PATCH DISEASE IN IRIDAEA-LAMINARIOIDES (RHODOPHYTA) CAUSED BY ENDOPHYTON SP (CHLOROPHYTA)
    (1994) CORREA, JA; FLORES, V; GARRIDO, J
    A green mottled coloration, or green patch disease, develops in the red alga Iridaea laminarioides Bory when it is infected by the algal endophyte Endophyton sp. The disease is widespread in host populations along the coast of central and southern Chile (33-degrees-17' S to 40-degrees-33' S) and affects both gametophytic and sporophytic fronds. It is characterized by green areas, usually located at the base of the thalli, which become soft in texture in fully developed infections. The softening of the host tissue is caused by cellular destruction resulting from endophyte-mediated compaction of the cells, followed by secondary bacterial infections. Bacteria gain access to the host through openings left in the host thallus during spore release from mature sporangia of Endophyton. Both life-history phases of L laminarioides were successfully infected by unialgal isolates of Endophyton in laboratory trials. Infection was achieved only by germlings from newly settled spores and the process of penetration was completed within 3 d. Softening of laboratory-infected thalli became evident about 8 wk after penetration and thallus destruction was complete after 3 to 4 mo of culture.
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    PEROXISOMAL ORGANIZATION IN NORMAL AND CEREBROHEPATORENAL (ZELLWEGER) SYNDROME FIBROBLASTS
    (1985) SANTOS, MJ; OJEDA, JM; GARRIDO, J; LEIGHTON, F
    The reported absence of morphologically detectable peroxisomes in liver and kidney tissue cells from patients affected by the autosomic recessive, inherited metabolic disease known as cerebrohepatorenal, or Zellweger, syndrome was studied in fibroblasts, assuming it to be a generalized defect. Normal cultured fibroblasts were shown to contain peroxisomes according to morphological, biochemical, and subcellular fractionation criteria: particle-bound catalase and fatty acyl-CoA oxidase copurify in subcellular fractionation by differential centrifugation or isopycnic equilibrium in continuous density gradients and peroxidase-positive organelles of .apprxeq. 0.1 .mu.m in diameter are detected in the cytoplasm. In Zellweger cultured fibroblasts, these peroxisomal enzymes are present; however, they behave as cytosolic enzymes in the different subcellular fractionation procedures employed and peroxisomes are not detected cytochemically. These findings support the hypothesis that the lack of peroxisomes in this genetic disease is the consequence of a defect in the assembly of the peroxisomal constituents. Furthermore, the value of fibroblasts for subcellular analysis of peroxisomal defects is illustrated.
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    PROTECTIVE ROLE OF BILIARY CHOLESTEROL AND PHOSPHOLIPID LAMELLAE AGAINST BILE ACID-INDUCED CELL-DAMAGE
    (W B SAUNDERS CO-ELSEVIER INC, 1994) PUGLIELLI, L; AMIGO, L; ARRESE, M; NUNEZ, L; RIGOTTI, A; GARRIDO, J; GONZALEZ, S; MINGRONE, G; GRECO, AV; ACCATINO, L; NERVI, F
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    STRUCTURE OF YEAST RNA-POLYMERASES DETERMINED BY ELECTRON-MICROSCOPY
    (1982) BULL, P; GARRIDO, J
    Methods were developed for the examination of yeast RNA polymerases I, II and III by EM. The size and shape of a eucaryotic RNA polymerase was established for the 1st time. The enzymes are roughly spherical in shape and compact in appearance. Their measured molecular diameters are 12.7 .+-. 0.4 and 11.0 .+-. 1.4 (SD) nm for polymerase I, 12.7 .+-. 1.1 and 12.2 .+-. 1.0 (SD) nm for polymerase II and 13.6 .+-. 0.6 and 11.5 .+-. 1.3 (SD) nm for polymerase III.
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    THE CYTOSKELETON OF GASTRIC-ACID SECRETING CELLS
    (1982) GARRIDO, J
    The role of the cytoskeleton in the process of acid secretion by oxyntic cells in briefly reviewed. In addition, it is shown that the typical disposition of cytoskeletal elements in the microvilli of the secretory pole of rat oxyntic cells is remarkably preserved after mild detergent extraction. This finding is discussed in terms of the usual model of filament-membrane interactions in microvilli.
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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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    THE ELECTRIC ORGAN OF DISCOPYGE-TSCHUDII - ITS INNERVATED FACE AND THE BIOLOGY OF ACETYLCHOLINESTERASE
    (1984) MENDEZ, B; GARRIDO, J; MALDONADO, M; JAKSIC, FM; INESTROSA, NC
    An ultrastructural, histochemical and biochemical study of the electric organ of the South American Torpedinid ray. D. tschudii was carried out. Fine structural cytochemical localization of acetylcholinesterase (AChE) indicated that most of the esterase was associated with the basal lamina. EM indicated no marked differences in the electrocyte ultrastructure between Discopyge and Torpedo californica. Discopyge electric organ possessed 3 molecular forms, 2 asymmetric forms (16 S and 13 S) and 1 globular hydrophobic form (6.5 S). The asymmetric 16 S AChE form was solubilized by heparin, a sulfated glycosaminoglycan, suggesting that heparin-like macromolecules are involved in the binding of the enzyme to the basal lamina. Cell-free translated AChE peptides, synthesized using Discopyge electric organ poly(A+) RNA, correspond to a main band of 62,000 daltons which probably represents the catalytic subunit of the asymmetric AChE.
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    ULTRACENTRIFUGAL ISOLATION OF VESICULAR CARRIERS OF BILIARY CHOLESTEROL IN NATIVE HUMAN AND RAT BILE
    (1987) ULLOA, N; GARRIDO, J; NERVI, F
    We have utilized ultracentrifugation of native bile-Metrizamide density gradients to isolate a vesicular transport system of biliary lipids in both man and rat. We identified vesicular structures by electron microscopy. Fresh bile specimens were obtained from bile fistula rats (unsaturated bile) and from patients 1 week after bile duct surgery (supersaturated bile). Metrizamide was dissolved in bile (33% w/v), and continuous density gradients were performed with undiluted bile (density limits = 1.020 to 1.300 gm per ml). The relative distribution of biliary cholesterol, phospholipid and bile salt was studied as a function of the density of the fractions. Approximately 50% of total rat biliary cholesterol and between 61 and 90% of human biliary cholesterol was concentrated in the lightest fractions of the gradients (density < 1.060 gm per ml). In contrast, less than 20% of bile salts was present in fractions with densities lwoer than 1.060 gm per ml. The highest amounts of bile salts and phospholipids of the bile-Metrizamide density gradients were found in the density range of 1.075 to 1.100 gm per ml in both human and rat bile. More than 80% of biliary proteins was found in fractions with densities > 1.075 gm per ml, and only 2% was found in cholesterol-rich fraction with density < 1.060 gm per ml in both species. When bile salt concentration was raised in rat bile from 38 to 97 mM by adding taurocholate, and low density cholesterol-rich fraction almost disappeared. Electron microscopy of negatively stained preparations of the fractions with density < 1.060 gm per ml showed 40 to 120 nm vesicles, which were not apparent in the other fractions. Similar vesicles were demonstrated also in fresh rat bile and within the canaliculi after acute depletion of the bile salt pool (biliary bile salt concentration of 3.45 mM; total biliary lipid concentration of 0.25 gm%). The structure of these vesicles was shown in thin sections of liver specimens. They appeared as internal cavities surrounded by a single, continuous 6-nm-thick bilayer. These studies demonstrate that a high proportion of biliary cholesterol is transported in vesicles in human supersaturated native bile and that vesicular carriers are also responsible for the transport of a significant amount of biliary cholesterol in unsaturated rat bile. The presence of vesicles in unsaturated hepatic bile strongly supports the thesis that biliary lipids may be secreted as vesicles from the hepatocyte into the canaliculi.