Browsing by Author "INESTROSA, NC"

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    A 13 KDA FRAGMENT IS RESPONSIBLE FOR THE HYDROPHOBIC AGGREGATION OF BRAIN G4 ACETYLCHOLINESTERASE
    (1988) FUENTES, ME; ROSENBERRY, TL; INESTROSA, NC
    Proteinase K treatment of the bovine brain acetylcholinesterase (AChE) releases a hydrophobic fragment of 13 kDa, which is entirely responsible entirely responsible for the aggregation of the G4 AChE in the absence of detergent. This observation provides evidence that the 13 kDa fragment, which comes from a previously identified 20 kDA subunit, is directly involved in the attachment of the G4 AChE to brain membranes. A model for the organization of the different sub-domains of the hydrophobic anchor of the G4 AChE is presented.
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    A COMPARISON OF THE XENOPUS-LAEVIS OOCYTE ACETYLCHOLINESTERASE WITH THE MUSCLE AND BRAIN ENZYME SUGGESTS VARIATIONS AT THE POSTTRANSLATIONAL LEVEL
    (1991) MOYA, MA; FUENTES, ME; INESTROSA, NC
    1. Xenopus laevis oocytes express endogenously two components of the cholinergic system: the muscarinic receptors and the acetylcholinesterase (AChE).
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    A HIGH-MOLECULAR-WEIGHT PROTEOGLYCAN IS DIFFERENTIALLY EXPRESSED DURING DEVELOPMENT OF THE MOLLUSK CONCHOLEPAS-CONCHOLEPAS (MOLLUSCA, GASTROPODA, MURICIDAE)
    (1992) BRANDAN, E; GONZALEZ, M; INESTROSA, NC; TREMBLAY, C; URREA, R
    Incorporation of radioactive sulfate to hatched veliger larvae of the gastropod muricid Concholepas concholepas indicated that over 87% of the sulfated macromolecules were found in the detergent insoluble fraction, rich in extracellular matrix (ECM) components. The sulfated material was solubilized with guanidine salt followed by urea dialysis and fractionated by DEAE-Sephacel chromatography. Three sulfated compounds eluting at 0.7, 1.1, and 3.0 M NaCl, called peaks I, II, and III, respectively, were obtained. The sulfated compound present in peak I was degraded by pronase or sodium alkaline treatment to a small sulfated resistant material, suggesting the presence of a proteoglycan (PG). Filtration analysis on Sephacryl S-500 and SDS-PAGE of the intact PG indicates that it has a high molecular weight (360,000 to over 1 X 10(6)). Monoclonal antibodies (mAb) against this PG were produced. The specificity of one mAb, the 6H2, was demonstrated by size chromatography and ELISA analysis. The epitope recognized by this mAb seems to be present in the core protein of the PG. Both the extent of sulfation and the presence of different sulfated species of PGs were evaluated during the development of this mollusc. A twelvefold increase in the incorporation of sulfate to PGs per milligram of protein was found in veliger larvae compared to blastula-glastula stages. This change correlated well with the differential expression of the sulfated PG present in peak I. Biochemical and immunological analysis indicate that high levels of this PG are found in veliger and trocophore larvae in comparison with blastula-gastrula and early juveniles. These results indicate that a high molecular weight PG probably of the ECM is differentially expressed during the development of the gastropod Concholepas concholepas.
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    A MEMBRANE-ASSOCIATED DIMER OF ACETYLCHOLINESTERASE FROM XENOPUS SKELETAL-MUSCLE IS SOLUBILIZED BY PHOSPHATIDYLINOSITOL-SPECIFIC PHOSPHOLIPASE-C
    (1988) INESTROSA, NC; FUENTES, ME; ANGLISTER, L; FUTERMAN, AH; SILMAN, I
    The susceptibility to phosphatidylinositol-specific phospholipase C of the membrane associated acetylcholinesterase (AChE) forms of Xenopus laevis skeletal muscle was examined. This treatment released almost all the detergent-soluble AChE species from muscle homogenates. Sucrose gradient analysis showed that the released acetylcholinesterase form corresponds to a hydrophilic G2 dimer, indicating that this dimer has a glycolipid anchoring domain which contains phosphatidylinositol.
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    A SIMPLE ASSAY TO ESTIMATE THE ACETYLCHOLINESTERASE MOLECULAR-FORMS IN CRUDE EXTRACTS OF RAT SKELETAL-MUSCLE
    (1989) PERELMAN, A; INESTROSA, NC
    All the current methods available for analyzing the acetylcholinesterase (AChE) molecular forms are time consuming and require the use of expensive equipment. We have found that by using the differential inactivation of globular (G4 + G1) and asymmetric AChE forms by high Mg2+ concentration, we can set up a very easy and quick assay that allows us to determine the relative proportions of AChE molecular forms present in rat skeletal muscles. This assay will be of great help in estimating changes in the muscle AChE forms under experimental conditions that require several simultaneous determinations.
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    ACETYLCHOLINESTERASE AGGREGATES IN A NEWLY FORMED MOTOR-NERVE SMOOTH-MUSCLE JUNCTION
    (1981) MENDEZ, B; INESTROSA, NC
    The changes in acetylcholinesterase (AChE) molecular forms were studied during cross-innervation of the inferior smooth muscle of the cat nictitating membrane by the hypoglossal nerve. One month after functional cross-innervation AChE activity increased by 2-fold above control values, and a new high MW AChE form (A12) was detected. BW284c51 [1,5-bis-(4-allyldimethylammoniumphenyl)-pentan-3-one dibromide], an anti-AChE, potentiated the contraction of the cross-innervated smooth muscle. AChE activity raised 6-fold above normal values 3 mo. later. At this time, half of the activity sediments to the bottom of the sucrose gradient and a time-dependent dissociation occurs in lighter AChE forms, reminiscent of AChE aggregates observed in the electric eel [Electrophorus electricus]. The multimolecular aggregates may be involved in the immobilization of AChE at the neuromuscular junction of a motor nerve and a smooth muscle.
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    ACETYLCHOLINESTERASE CHANGES IN HEARTS WITH SINUS RHYTHM AND ATRIAL-FIBRILLATION
    (1993) GONZALEZ, RA; CAMPOS, EO; KARMELIC, C; MORAN, S; INESTROSA, NC
    1. Clinical and experimental evidence suggest that changes in the autonomic tone play a role in the pathogenesis of atrial fibrillation.
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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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    ACYL-COA SYNTHETASE AND THE PEROXISOMAL ENZYMES OF BETA-OXIDATION IN HUMAN-LIVER - QUANTITATIVE-ANALYSIS OF THEIR SUBCELLULAR-LOCALIZATION
    (1984) BRONFMAN, M; INESTROSA, NC; NERVI, FO; LEIGHTON, F
    The presence of acyl CoA synthetase (EC 6.2.1.3) in peroxisomes and the subcellular distribution of .beta.-oxidation enzymes in human liver were investigated by using a single-step fractionation method of whole liver homogenates in metrizamide continuous density gradients and a novel procedure of computer analysis of results. Peroxisomes contain 16% of the liver palmitoyl CoA synthetase activity, and 21% and 60% of the enzyme activity was localized in mitochondria and microsomal fractions, respectively. Fatty acyl CoA oxidase was localized exclusively in peroxisomes, confirming previous results. Human liver peroxisomes contribute 13%, 17% and 11% of the liver activities of crotonase, .beta.-hydroxyacyl CoA dehdyrogenase and thiolase, respectively. The absolute activities found in peroxisomes for the enzymes investigated suggest that in human liver fatty acyl CoA oxidase is the rate-limiting enzyme of the peroxisomal .beta.-oxidation pathway, when palmitic acid is the substrate.
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    AGE-RELATED RESPONSES OF SKELETAL-MUSCLE AFTER ECTOPIC INNERVATION, WITH PARTICULAR REFERENCE TO 16S ACETYLCHOLINESTERASE, IN ADULT-RATS
    (1984) MALDONADO, M; RAMIREZ, BU; RUIZ, G; YAMUY, J; INESTROSA, NC
    The formation of ectopic junctions between the foreign fibular nerve and the soleus muscle of young (35-day-old) and mature (200-day-old) adult rats was induced by severing the normal nerve 4 wk after implanting the foreign nerve. The various molecular forms of acetylcholinesterase (AChE) were studied both at the implanted region and at the original denervated endplates. The velocity of contraction was also studied. In young rats the 16S form was first detected in the ectopic junctions around day 5 after reinnervation; this form rapidly increased during the following weeks, reaching a plateau by day 20. By contrast, in mature rats the appearance of the 16S AChE was dramatically delayed; in fact, it could not be observed before day 80 after reinnervation. (The 16S AChE form appeared at day 20 after reinnervation in the original denervated endplates of young rats; however, at the same time, no effect was observed in mature animals.) The original, slow muscle fibers of the soleus became faster upon reinnervation; this change also occurred much earlier in younger than in mature rats. A loss of plasticity in the skeletal muscle of mature rats is indicated. Caution is suggested in the use of the ectopic innervation model to study development in mature adult rats.
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    AMPHIPHILIC BEHAVIOR OF A BRAIN TETRAMERIC ACETYLCHOLINESTERASE FORM LACKING THE PLASMA-MEMBRANE ANCHORING DOMAIN
    (1992) FUENTES, ME; INESTROSA, NC
    We have studied the behavior of a mammalian brain tetrameric acetylcholinesterase (AChE) form released by proteinase K from a crude membrane fraction of bovine caudate nucleus. The solubilization of active AChE indicated the presence of a protease-sensitive site in the anchored protein. Unexpectedly, the solubilized AChE maintained its capacity to form aggregates in detergent-free gradients. We demonstrate here that this property was due neither to the presence of the hydrophobic membrane-anchoring domain still linked to the enzyme, nor to the presence of AChE activity trapped in small plasma membrane vesicles. Moreover, we found that the proteinase K-treated extract, devoid of AChE activity, induced the aggregation of purified hydrophilic AChE which usually does not form aggregates. Our results suggest the presence of an AChE aggregating factor in bovine brain extracts prepared in the presence of proteinase K. It is possible that this aggregation may reflect a process of AChE clustering on neurons.
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    ANTI-200 KDA NEUROFILAMENT ANTIBODY CROSS-REACTS WITH MICROTUBULE-ASSOCIATED PROTEIN-2 (MAP-2)
    (1989) ALLENDE, ML; KRAUSS, RY; TREMBLAY, C; ALVAREZ, J; INESTROSA, NC
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    AXONAL SPROUTING INDUCED IN THE SCIATIC-NERVE BY THE AMYLOID PRECURSOR PROTEIN (APP) AND OTHER ANTIPROTEASES
    (1992) ALVAREZ, J; MORENO, RD; LLANOS, O; INESTROSA, NC; BRANDAN, E; COLBY, T; ESCH, FS
    Protease inhibition is the mechanism by which some trophic factors promote the extension of neurites. In the rat sciatic nerve, we assessed the ability to induce sprouts of the APP isoform that embodies the Kunitz antiprotease domain and other antiproteases. With the electron microscope, axonal sprouts were found when antiproteases were supplied but not after administration of inactive substances. We conclude that axons have a drive to sprout which can be released by the unbalance of an extracellular protease antiprotease system. We propose that this system is involved in the pathogenesis of Alzheimer's disease.
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    AXONS GROW IN THE AGING RAT BUT FAST TRANSPORT AND ACETYLCHOLINESTERASE CONTENT REMAIN UNCHANGED
    (1988) INESTROSA, NC; ALVAREZ, J
    Caliber and microtubular density of myelinated fibers, acetylcholinesterase (AChE) content and its accumulation at a ligature were studied in the phrenic nerve of mature (3-4 months) and aging (2-year-old) rats. The number of axons remained constant. The cross-sectional area of the nerve was 67% greater in the older group: the axoplasm, though, constituted about 20% of the nerve tissue irrespective of age. The mean cross-sectional area of myelinated axons was twice as big in aging compared to mature rats. All axons grew in the same proportion irrespective of their original caliber. The microtubular density of 3-.mu.m axons was about 22 microtubules .mu.m2 in mature and aging rats. The AChE activity of aging rats was half as much as that of mature rats if it was expressed per wet weight of nerve tissue but did not change if it was expressed per nerve fiber. Twenty-four hours after ligation of the nerve, total AChE activity rose in mature and aging rats by ca. 168%: the molecular forms.sbd.asymmetric and globular.sbd.accumulated in the same proportion in both age groups. We conclude that myelinated axons grow in the adult stage of life but the structure of axoplasm, content of AChE per axon, and rate of fast transport remain lifelong features of nerve fibers.
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    BINDING OF THE ASYMMETRIC FORMS OF ACETYLCHOLINESTERASE TO HEPARIN
    (1984) BRANDAN, E; INESTROSA, NC
    The interaction between acetylcholinesterase (EC 3.1.1.7) and heparin, a sulfated glycosaminoglycan, was studied by affinity chromatography. A specific binding of the asymmetric acetylcholinesterase to an agarose gel containing covalently bound heparin was demonstrated. This interaction required an intact collagenous tail, shown by the fact that the binding is abolished by pretreatment with collagenase. Globular forms did not bind to the column. Total and intracellular asymmetric acetylcholinesterase forms isolated from the endplate region of the rat diaphragm muscle showed higher affinity for the heparin than did the enzyme from the non-endplate region. Binding to the resin was destabilized with 0.55 M NaCl, and, among the various glycosaminoglycans tested, only heparin was able to displace the acetylcholinesterase bound to the column. Apparently, asymmetric acetylcholinesterase forms are immobilized on the synaptic basal lamina via interactions with heparin-like molecules, probably related to heparan sulfate proteoglycans.
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    BIOSYNTHESIS OF THE NEUROFILAMENT HEAVY SUBUNIT IN XENOPUS OOCYTES MICROINJECTED WITH RAT-BRAIN POLY(A)+ RNA
    (1987) CROSS, D; ALLENDE, ML; KRAUSS, RY; FUENTES, ME; KALTWASSER, G; ALVAREZ, J; INESTROSA, NC
    In order to study the expression of the major subunit of neurofilaments (NFs), rat brain poly(A)+ RNA was purified by three different procedures and was injected in Xenopus laevis oocytes. This system was able to translate efficiently the 200 kDa NF subunit as shown by a dot-blot immunoassay and by immunoprecipitation of labeled NF polypeptides.
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    BLOOD MARKERS IN ALZHEIMER-DISEASE - SUBNORMAL ACETYLCHOLINESTERASE AND BUTYRYLCHOLINESTERASE IN LYMPHOCYTES AND ERYTHROCYTES
    (ELSEVIER SCIENCE BV, 1994) INESTROSA, NC; ALARCON, R; ARRIAGADA, J; DONOSO, A; ALVAREZ, J; CAMPOS, EO
    In patients with the clinical diagnosis of Alzheimer disease (AD), we searched for systemic changes in components of the blood as a diagnostic tool. The acetylcholine-related enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) were measured in plasma, erythrocytes, platelets and lymphocytes. Results did not show a general effect; notwithstanding, specific cell types presented alterations either in AChE or BuChE but not in both enzymatic activities. In AD patients, AChE of lymphocytes was reduced by 60% compared with the age-matched controls. However, when patients were divided, the sporadic but not the familial subgroup exhibited a significant reduction. In erythrocytes the BuChE activity was reduced by 45% in sporadic AD. The molecular forms of the lymphocyte AChE were characterized by velocity sedimentation. Both globular forms were subnormal, more so the tetrameric G(4) AChE form than the G(2) form.
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    CHANGES IN CONTRALATERAL SYNAPTIC ACETYLCHOLINESTERASE FOLLOWING MOTOR-NERVE SECTION IN RATS
    (1988) FADIC, R; SOZA, MA; INESTROSA, NC
    We report here that denervation of rat extensor digitorum longus, soleus and diaphragm muscle results in an increase of a subset of asymmetric acetylcholinesterase (class II A-forms) in the contralateral muscle, within a few days. This observation is interesting because it suggests a specific regulation of one asymmetric enzyme fraction, which is solubilized only in the presence of chelating agents and is thought to reside in the basal lamina.
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    CHARACTERIZATION OF A TETRAMERIC G4 FORM OF ACETYLCHOLINESTERASE FROM BOVINE BRAIN - A COMPARISON WITH THE DIMERIC G2 FORM OF THE ELECTRIC ORGAN
    (1988) FUENTES, ME; INESTROSA, NC
    Globular forms (G forms) of acetylcholinesterase (AChE) are formed by monomers, dimers and tetramers of the catalytic subunits (G1, G2 and G4). In this work the hydrophobic G2 and G4 AChE forms were purified to homogeneity from Discopyge electric organ and bovine caudate nucleus and studied from different points of view, including: velocity sedimentation, affinity to lectins and SDS-polyacrylamiide gel electrophoresis under reducing and non-reducing conditions. The polypeptide composition of Discopyge electric organ G2 is similar to Torpedo, however the pattern of the brain G4 AChE is much complex. Under non-reducing conditions the catalytic subunit possesses a molecular weight of 65 kDa, however this value increases to 68 kDa after reduction, suggesting that intrachain-disulfide bonds are important in the folding of the catalytic subunits of the AChE. Also it was found that after mild proteolysis; the (125I)-TID-20 kDa fragment decreased its molecular weight to approximately 10 kDa with little loss of AChE activity. Finally, we suggest a model for the organization of the different domains of the hydrophobic anchor fragment of the G4 form.