34 results on '"Madeddu L"'
Search Results
2. Specific Localization of the α-Latrotoxin Receptor in the Nerve Terminal Plasma Membrane
- Author
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Valtorta, F., Madeddu, L., Meldolesi, J., and Ceccarelli, B.
- Published
- 1984
3. Characterization of Multigene Families in the Micronuclear Genome of Paramecium Tetraurelia Reveals a Germline Specific Sequence in an Intron of a Centrin Gene
- Author
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Vayssie, L., primary, Sperling, L., additional, and Madeddu, L., additional
- Published
- 1997
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4. A large multigene family codes for the polypeptides of the crystalline trichocyst matrix in Paramecium.
- Author
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Madeddu, L, primary, Gautier, M C, additional, Vayssié, L, additional, Houari, A, additional, and Sperling, L, additional
- Published
- 1995
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5. Protein processing and morphogenesis of Paramecium secretory granules
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Gautier, M.-C., primary, Madeddu, L., additional, Loubresse, Garreau N., additional, and Sperling, L., additional
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- 1995
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6. Evidence for defects in membrane traffic in Paramecium secretory mutants unable to produce functional storage granules
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Gautier, MC, primary, Garreau de Loubresse, N, additional, Madeddu, L, additional, and Sperling, L, additional
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- 1994
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7. Protein processing and morphogenesis of secretory granules in Paramecium
- Author
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Madeddu, L., primary, Gautier, M.C., additional, Le Caer, J.P., additional, de Loubresse, N.Garreau, additional, and Sperling, L., additional
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- 1994
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8. Maturation of dense core granules in wild type and mutant Tetrahymena thermophila.
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Turkewitz, A.P., primary, Madeddu, L., additional, and Kelly, R.B., additional
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- 1991
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9. Intracellular Calcium Homeostasis in a Human Neuroblastoma Cell Line: Modulation by Depolarization, Cholinergic Receptors, and α-Latrotoxin.
- Author
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Sher, E., Gotti, C., Pandiella, A., Madeddu, L., and Clementi, F.
- Published
- 1988
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10. Specific localization of the alpha-latrotoxin receptor in the nerve terminal plasma membrane.
- Author
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Valtorta, F, Madeddu, L, Meldolesi, J, and Ceccarelli, B
- Abstract
The receptor for alpha-latrotoxin, the major protein component of the black widow spider venom, was investigated by the use of the purified toxin and of polyclonal, monospecific anti-alpha-latrotoxin antibodies. Experiments on rat brain synaptosomes (where the existence of alpha-latrotoxin receptors was known from previous studies) demonstrated that the toxin-receptor complex is made stable by glutaraldehyde fixation. At saturation, each such complex was found to bind on the average five antitoxin antibody molecules. In frog cutaneous pectoris muscles, the existence of a finite number of high-affinity receptors was revealed by binding experiments with 125I-alpha-latrotoxin (Kd = 5 X 10(-10) M; bmax = 1.36 +/- 0.16 [SE] X 10(9) sites/mg tissue, dry weight). Nonpermeabilized muscles were first treated with alpha-latrotoxin, and then washed, fixed, dissociated into individual fibers, and treated with anti-alpha-latrotoxin antibodies and finally with rhodamine-conjugated sheep anti-rabbit antibodies. In these preparations, muscle fibers and unmyelinated preterminal nerve branches were consistently negative, whereas bright specific fluorescent images, indicative of concentrated alpha-latrotoxin binding sites, appeared in the junctional region. These images closely correspond in size, shape, and localization to endplates decorated by the acetylcholinesterase reaction. The presynaptic localization of the specific fluorescence found at frog neuromuscular junctions is supported by two sets of findings: (a) fluorescent endplate images were not seen in muscles that had been denervated; and (b) the distribution of fluorescence in many fibers treated with alpha-latrotoxin at room temperature was the one expected from swollen terminal branches. Swelling of terminals is a known morphological change induced by alpha-latrotoxin in this preparation. When muscles were treated with either proteolytic enzymes (trypsin, collagenase) or detergents (Triton X-100) before exposure to alpha-latrotoxin, the specific fluorescent endplate images failed to appear. Taken together these findings indicate that the alpha-latrotoxin receptor is an externally exposed protein highly concentrated in the nerve terminal plasma membrane. Its density (number per unit area) at the frog neuromuscular junction can be calculated to be approximately 2,400/micron2.
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- 1984
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11. Second-messenger generation in PC12 cells. Interactions between cyclic AMP and Ca2+ signals
- Author
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Gatti, G, Madeddu, L, Pandiella, A, Pozzan, T, and Meldolesi, J
- Abstract
Changes in cyclic AMP concentrations were studied in intact PC12 pheochromocytoma cells exposed to a variety of treatments. A marked increase was triggered by N-(L-2-phenylisopropyl)adenosine, the activator of an adenosine receptor, whereas a decrease (observed even after phosphodiesterase blockade) was induced by carbachol, working through a muscarinic receptor inhibited by the selective muscarinic blocker pirenzepine, only at high concentration (Ki 450 nM). A decrease in cyclic AMP was also induced by clonidine, an alpha 2-adrenergic-receptor agonist. Both the alpha 2-adrenergic and the muscarinic inhibitions were prevented by pretreatment of the cells with pertussis toxin, and were unaffected by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate. The latter drug caused a decrease in the resting cyclic AMP concentrations, and a potentiation of the increase induced by adenosine-receptor activation. Except for clonidine, all these treatments were found to be effective in both growing PC12 cells and, although to a smaller degree, in cells that had stopped growing and had acquired a neuron-like phenotype after prolonged treatment with nerve growth factor (NGF). Neither forskolin (a direct activator of adenylate cyclase) nor the activation of adenosine and alpha-adrenergic receptors was able to modify the resting cytosolic Ca2+ concentration [Ca2+]i in PC12 cells. Likewise, the K+-induced [Ca2+]i transients were unchanged after these treatments, whereas the transients induced by carbachol through the activation of a muscarinic receptor highly sensitive to pirenzepine were moderately potentiated by forskolin (and, to a lesser degree, by the adenosine analogue) and attenuated by clonidine. These results characterize in further detail the spectrum and the mutual interrelationships of the intracellular signals induced by receptor activation in PC12 cells, also as a function of the NGF-induced differentiation.
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- 1988
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12. Mode of action of alpha-latrotoxin: role of divalent cations in Ca2(+)-dependent and Ca2(+)-independent effects mediated by the toxin.
- Author
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Rosenthal, L, Zacchetti, D, Madeddu, L, and Meldolesi, J
- Abstract
The potent neurotoxin alpha-latrotoxin (alpha LTx), from black widow spider venom, induces neurotransmitter release in both Ca2(+)-containing and Ca2(+)-free medium, following interaction with a specific cell surface receptor. Binding studies revealed two populations of alpha LTx binding sites in bovine synaptosomal membranes, showing the same high affinity (Kd, 0.3 x 10(-10) M) for alpha LTx, with approximately 50% of the sites being Ca2+ sensitive and the rest being Ca2+ insensitive. In contrast, in PC12 cells alpha LTx binding was completely unaffected by the removal of extracellular Ca2+ (Kd, 5 x 10(-10) M). The use of La3+ as an inhibitor of alpha LTx action, previously shown in synaptosomes, was extended to PC12 cells. In this system, La3+ (100 microM) was shown to inhibit Ca2+ influx, both Ca2(+)-dependent and -independent dopamine release, and polyphosphoinositide (PPI) hydrolysis induced by alpha LTx. At the same time, La3+ did not block alpha LTx binding or dopamine release evoked by either the ionophore ionomycin (0.5 microM) or the phorbol ester tetradecanoylphorbol acetate (100 nM). La3+ also blocked the influx of Mn2+ ions through the alpha LTx-induced cation channel, as measured by quenching of fura-2 fluorescence. In this PC12 cell line, PPI hydrolysis could also be induced by ionomycin, but only when it was present at concentrations that caused an elevation of free intracellular Ca2+ ([Ca2+]i) that was not transient but was as persistent as that evoked by alpha LTx. Our conclusions with regard to the mode of action of alpha LTx are as follows. (i) All the effects of alpha LTx in PC12 cells (dopamine release, PPI hydrolysis, and Ca2+ influx) can be mediated via a single, Ca2(+)-insensitive alpha LTx receptor. (ii) alpha LTx-induced PPI hydrolysis is most likely due to the activation of a Ca2(+)-sensitive phospholipase C following the persistent rise in [Ca2+]i elicited by the toxin in Ca2(+)-containing medium, and not via direct coupling of the alpha LTx receptor to the enzyme. (iii) Toxin-evoked Ca2(+)-independent dopamine release can be blocked by La3+ at the extracellular level, most likely by prevention of the entry of divalent cations.
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- 1990
13. Black widow spider venom-induced release of neurotransmitters: Mammalian synaptosomes are stimulated by a unique venom component (α-Latrotoxin), insect synaptosomes by multiple components
- Author
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Knipper, M., primary, Madeddu, L., additional, Breer, H., additional, and Meldolesi, J., additional
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- 1986
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14. α-Latrotoxin and glycerotoxin differ in target specificity and in the mechanism of their neurotransmitter releasing action
- Author
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Madeddu, L., primary, Meldolesi, J., additional, Pozzan, T., additional, Sanclemente, L.E. Cardona, additional, and Bon, C., additional
- Published
- 1984
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15. The effect of α-latrotoxin on the neurosecretory PC12 cell line: Studies on toxin binding and stimulation of transmitter release
- Author
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Meldolesi, J., primary, Madeddu, L., additional, Torda, M., additional, Gatti, G., additional, and Niutta, E., additional
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- 1983
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16. Assessment of community efforts to advance network-based prediction of protein-protein interactions.
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Wang XW, Madeddu L, Spirohn K, Martini L, Fazzone A, Becchetti L, Wytock TP, Kovács IA, Balogh OM, Benczik B, Pétervári M, Ágg B, Ferdinandy P, Vulliard L, Menche J, Colonnese S, Petti M, Scarano G, Cuomo F, Hao T, Laval F, Willems L, Twizere JC, Vidal M, Calderwood MA, Petrillo E, Barabási AL, Silverman EK, Loscalzo J, Velardi P, and Liu YY
- Subjects
- Animals, Humans, Caenorhabditis elegans, Protein Interaction Maps, Computational Biology methods, Protein Interaction Mapping methods, Saccharomyces cerevisiae
- Abstract
Comprehensive understanding of the human protein-protein interaction (PPI) network, aka the human interactome, can provide important insights into the molecular mechanisms of complex biological processes and diseases. Despite the remarkable experimental efforts undertaken to date to determine the structure of the human interactome, many PPIs remain unmapped. Computational approaches, especially network-based methods, can facilitate the identification of previously uncharacterized PPIs. Many such methods have been proposed. Yet, a systematic evaluation of existing network-based methods in predicting PPIs is still lacking. Here, we report community efforts initiated by the International Network Medicine Consortium to benchmark the ability of 26 representative network-based methods to predict PPIs across six different interactomes of four different organisms: A. thaliana, C. elegans, S. cerevisiae, and H. sapiens. Through extensive computational and experimental validations, we found that advanced similarity-based methods, which leverage the underlying network characteristics of PPIs, show superior performance over other general link prediction methods in the interactomes we considered., (© 2023. The Author(s).)
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- 2023
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17. A Network-Based Analysis of Disease Modules From a Taxonomic Perspective.
- Author
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Grani G, Madeddu L, and Velardi P
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- Humans, Reproducibility of Results, Algorithms
- Abstract
Objective: Human-curated diseaseontologies are widely used for diagnostic evaluation, treatment and data comparisons over time, and clinical decision support. The classification principles underlying these ontologies are guided by the analysis of observable pathological similarities between disorders, often based on anatomical or histological principles. Although, thanks to recent advances in molecular biology, disease ontologies are slowly changing to integrate the etiological and genetic origins of diseases, nosology still reflects this "reductionist" perspective. Proximity relationships of disease modules (hereafter DMs) in the human interactome network are now increasingly used in diagnostics, to identify pathobiologically similar diseases and to support drug repurposing and discovery. On the other hand, similarity relations induced from structural proximity of DMs also have several limitations, such as incomplete knowledge of disease-gene relationships and reliability of clinical trials to assess their validity. The purpose of the study described in this paper is to shed more light on disease similarities by analyzing the relationship between categorical proximity of diseases in human-curated ontologies and structural proximity of the related DMs in the interactome., Method: We propose a method (and related algorithms) to automatically induce a hierarchical structure from proximity relations between DMs, and to compare this structure with a human-curated disease taxonomy., Results: We demonstrate that the proposed method allows to systematically analyze commonalities and differences among structural and categorical similarity of human diseases, help refine and extend human disease classification systems, and identify promising network areas where new disease-gene interactions can be discovered.
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- 2022
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18. Integrating categorical and structural proximity in Disease Ontologies.
- Author
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Madeddu L, Grani G, and Velardi P
- Subjects
- Humans, Algorithms
- Abstract
The purpose of the study described in this paper is to shed more light on disease similarities by analyzing the relationship between categorical proximity of diseases in human-curated ontologies and structural proximity of the related disease module (DM) in the interactome. We propose a methodology (and related algorithms) to automatically induce a hierarchical structure from proximity relations between DMs, and to compare this structure with a human-curated disease taxonomy.Clinical relevance- Disease ontologies are extensively used for diagnostic evaluation and clinical decision support but still reflect the clinical reductionist perspective. We demonstrate that the proposed network-based methodology allows us to analyze commonalities and differences among structural and categorical similarity of human diseases, help refine human disease classification systems, and identify promising network areas where new disease-gene interactions can be discovered.
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- 2021
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19. Homology-dependent gene silencing in Paramecium.
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Ruiz F, Vayssié L, Klotz C, Sperling L, and Madeddu L
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- Animals, Cilia chemistry, Exocytosis, Gene Dosage, Membrane Proteins genetics, Microinjections, Mutation, Paramecium physiology, Phenotype, Plasmids, Protozoan Proteins metabolism, Gene Expression Regulation, Genes, Protozoan, Paramecium genetics, Protozoan Proteins genetics
- Abstract
Microinjection at high copy number of plasmids containing only the coding region of a gene into the Paramecium somatic macronucleus led to a marked reduction in the expression of the corresponding endogenous gene(s). The silencing effect, which is stably maintained throughout vegetative growth, has been observed for all Paramecium genes examined so far: a single-copy gene (ND7), as well as members of multigene families (centrin genes and trichocyst matrix protein genes) in which all closely related paralogous genes appeared to be affected. This phenomenon may be related to posttranscriptional gene silencing in transgenic plants and quelling in Neurospora and allows the efficient creation of specific mutant phenotypes thus providing a potentially powerful tool to study gene function in Paramecium. For the two multigene families that encode proteins that coassemble to build up complex subcellular structures the analysis presented herein provides the first experimental evidence that the members of these gene families are not functionally redundant.
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- 1998
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20. Characterization of centrin genes in Paramecium.
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Madeddu L, Klotz C, Le Caer JP, and Beisson J
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- Amino Acid Sequence, Animals, Antibodies, Protozoan, Base Sequence, Calcium-Binding Proteins immunology, Ciliophora chemistry, Ciliophora immunology, Cloning, Molecular, Conserved Sequence, Contractile Proteins immunology, Gene Expression, Molecular Sequence Data, Paramecium ultrastructure, Sequence Analysis, DNA, Sequence Homology, Amino Acid, Calcium-Binding Proteins genetics, Chromosomal Proteins, Non-Histone, Contractile Proteins genetics, Fungal Proteins chemistry, Fungal Proteins genetics, Isocitrate Lyase chemistry, Isocitrate Lyase genetics, Paramecium genetics, Protozoan Proteins chemistry, Protozoan Proteins genetics
- Abstract
Centrins are highly conserved, ubiquitous cytoskeletal components which belong to the EF-hand superfamily of Ca2+-modulated proteins. We report here the molecular characterization of new members of the centrin family, Paramecium centrins. Previous studies described the organization of the infraciliary lattice (ICL), the innermost cortical cytoskeletal network of Paramecium, and showed that it was composed of a set of low-molecular-mass, Ca2+-binding polypeptides [Garreau de Loubresse, N., Klotz, C., Vigues, B., Rutin, J & Beisson, J. (1991) Biol. Cell 71, 217-225]. In this paper we show that these polypeptides are recognized by specific anti-centrin polyclonal antibodies. Their microsequences revealed four distinct N-termini. For one of them, ICL1, N-terminal and internal peptide sequences were used for PCR amplification and cloning of a DNA fragment containing a complete centrin coding sequence. The deduced amino acid sequence presents about 50% identify with those of centrins from other species. Further molecular analysis allowed us to identify two additional closely related, co-expressed ICL1 genes, providing the first example of a centrin multigenic family in a microorganism.
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- 1996
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21. Palliative home care and place of death among cancer patients: a population-based study.
- Author
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Costantini M, Camoirano E, Madeddu L, Bruzzi P, Verganelli E, and Henriquet F
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- Age Factors, Aged, Aged, 80 and over, Educational Status, Female, Humans, Italy epidemiology, Male, Middle Aged, Multivariate Analysis, Population Surveillance, Home Care Services statistics & numerical data, Hospitalization, Neoplasms mortality, Palliative Care
- Abstract
This population-based study of all cancer deaths (n = 12,343) occurring in Genoa, Italy, from 1986 to 1990 investigated the relation between place of death and age, sex, marital status, education, cancer site and provision of palliative home care (PHC). The proportion of home deaths significantly increased from 27.9% (1986) to 33.0% (1990) and was twice as frequent among PHC users (60.8%) than among nonusers (29.3%). The number of patients dying of cancer who received PHC increased from 41 in 1986 (1.6% of cancer deaths) to 191 in 1990 (8.0% of cancer deaths). PHC users, when compared to nonusers were younger, more frequently married, had a higher level of education and were more frequently affected by cancers of the lung, breast or prostate. Multivariate analysis shows that the probability of home death increased with increasing age and education level and was higher in females and in married patients. The provision of PHC was the strongest predictor of home death (OR = 4.00; 95% CI = 3.33-4.81), while the temporal trend almost disappeared. These results suggest that most of the increase in home deaths from 1986 to 1990 is attributable to the PHC and that expansion of the PHC services may enable about 60% of cancer patients to die at home. These results appear to be desirable from the individual patient's viewpoint and in a public health perspective.
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- 1993
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22. Intracellular Ca2+ storage organelles in non-muscle cells: heterogeneity and functional assignment.
- Author
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Meldolesi J, Madeddu L, and Pozzan T
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- Animals, Microsomes metabolism, Mitochondria metabolism, Calcium metabolism, Organelles metabolism
- Published
- 1990
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23. Calsequestrin, a component of the inositol 1,4,5-trisphosphate-sensitive Ca2+ store of chicken cerebellum.
- Author
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Volpe P, Alderson-Lang BH, Madeddu L, Damiani E, Collins JH, and Margreth A
- Subjects
- Animals, Calcium-Transporting ATPases metabolism, Calsequestrin chemistry, Chickens, Immunologic Techniques, Microsomes metabolism, Peptide Mapping, Tissue Distribution, Calcium metabolism, Calsequestrin metabolism, Cerebellum metabolism, Inositol 1,4,5-Trisphosphate pharmacology
- Abstract
The presence and distribution of calsequestrin (CS), Ca2+ pump, and inositol 1,4,5-trisphosphate (IP3) receptor were investigated biochemically and immunologically in microsomal (P3) fractions isolated from chicken cerebrum and cerebellum. Two different batches of polyclonal antibodies specific for chicken skeletal muscle CS identified a Ca2+ binding, CS-like protein that was extremely enriched in cerebellum P3 fractions and absent from all cerebrum fractions. The cerebellum CS-like protein was deemed authentic CS because the N-terminal amino acid domain and peptide mapping were identical to those of skeletal muscle CS in the same species. CS was detected in striated muscles and cerebellum only. Cerebellum P3 fractions were also found to be considerably enriched in Ca2+ pump and IP3 receptor compared with the homologous cerebrum fractions, as judged by measurements of Ca2+ uptake, Ca2(+)-ATPase activity, IP3-induced Ca2+ release, and [3H]IP3 binding, respectively. Cerebellum microsomal fractions therefore appear to contain membrane fragments endowed with Ca2+ pump, IP3 receptor, and CS, i.e., three key components of a Ca2+ storage organelle.
- Published
- 1990
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24. Alpha-latrotoxin and glycerotoxin differ in target specificity and in the mechanism of their neurotransmitter releasing action.
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Madeddu L, Meldolesi J, Pozzan T, Cardona Sanclemente LE, and Bon C
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- Animals, Calcium metabolism, Cell Line, Cerebral Cortex metabolism, Corpus Striatum metabolism, Electric Organ metabolism, Helminth Proteins, In Vitro Techniques, Snake Venoms, Synaptosomes metabolism, Venoms, Arthropod Venoms pharmacology, Neurotoxins pharmacology, Neurotransmitter Agents metabolism, Spider Venoms pharmacology, Synaptic Membranes drug effects
- Abstract
alpha-Latrotoxin, a high molecular weight protein (130,000) purified from the venom of the black widow spider, and a partially purified neurotoxin, glycerotoxin, prepared from extracts of the jaw glands of the polichaete annelid Glycera convoluta, were previously found to induce similar effects (stimulation of quantal acetylcholine release) at the frog neuromuscular junction. In the present study parallel experiments performed with these two toxins revealed that only glycerotoxin was able to release acetylcholine from Torpedo electric organ synaptosomes, while alpha-latrotoxin did not affect release in this system. In contrast, alpha-latrotoxin stimulated release of dopamine from PC12 cells (a cloned neurosecretory cell line), whereas glycerotoxin was almost inactive. In rat brain synaptosomes both toxins were active. Preincubation of synaptosomal membranes with glycerotoxin was without effect on the subsequent binding of alpha-latrotoxin. Glycerotoxin application induced depolarization of synaptosomal plasma membrane, massive Ca2+ influx, marked increase of the cytosolic Ca2+ concentration, and stimulation of catecholamine release. The latter effect occurred to the same extent when glycerotoxin was applied either in complete medium (containing both Ca2+ and Mg2+), Ca2+-free medium or divalent cation-free medium. Some of these effects of glycerotoxin in rat brain synaptosomes (depolarization, increased Ca2+ influx and increased cytosolic Ca2+ concentration) resemble effects previously reported for alpha-latrotoxin. However, the secretory response induced by the latter was reduced in Ca2+-free, and abolished in divalent cation-free media. The different target specificity and the lack of binding competition of the two toxins could be due to their ability to recognize different receptors whose distribution overlap only in part in the cellular systems we have studied. The differences in action, on the other hand, could depend on postreceptor events, possibly related to the transmembrane insertion of toxin molecules demonstrated by others in artificial lipid membranes.
- Published
- 1984
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25. Alpha latrotoxin of black widow spider venom: an interesting neurotoxin and a tool for investigating the process of neurotransmitter release.
- Author
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Scheer H, Madeddu L, Dozio N, Gatti G, Vicentini LM, and Meldolesi J
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- Animals, Cations, Divalent metabolism, Ion Channels drug effects, Ion Channels metabolism, Neuromuscular Junction drug effects, Phosphatidylinositols metabolism, Protein Kinase C, Protein Kinases metabolism, Receptors, Cholinergic metabolism, Arthropod Venoms pharmacology, Neurotoxins pharmacology, Neurotransmitter Agents metabolism, Receptors, Peptide, Spider Venoms pharmacology
- Abstract
Alpha latrotoxin, purified from the venom of the black widow spider, is a high Mr (130000) protein devoid of detectable enzymatic activity. When applied to vertebrate nerve terminals (of the central as well as peripheral nervous systems) the toxin elicits massive release of neurotransmitters by stimulating the fusion of synaptic vesicles with the presynaptic membrane (exocytosis). Among non-neuronal systems, only the neurosecretory cell line PC12 is sensitive to alpha latrotoxin; all others investigated so far are insensitive. In order to act, alpha latrotoxin requires the presence of divalent cations in the medium. Ca2+ can be substituted by other divalent cations as Sr2+, Ba2+, Mn2+, Mg2+. However, with the last two the catecholamine release response is reduced in PC12 cells and synaptosomes. A specific, high affinity receptor of alpha latrotoxin exists in preparation sensitive to the toxin. This receptor has been purified and found to be a high Mr, integral membrane protein. In the frog neuromuscular junction the receptor is localized exclusively in the presynaptic membrane. Binding of alpha latrotoxin to the receptor in a Ca2+-containing incubation medium induces membrane depolarization (insensitive to tetrodotoxin), stimulation of Ca2+ influx (insensitive to verapamil) with consequent increase in the cytoplasmic free Ca2+ concentration and stimulation of phosphoinositide breakdown. In Ca2+ free medium depolarization is maintained, but free Ca2+ concentration does not rise after toxin application. In conclusion, alpha latrotoxin seems to act through a dual mechanism. The Ca2+-independent part of this mechanism may be mediated by the activation of protein kinase C, triggered by phosphoinositide metabolism. The relevance of these findings for presynaptic physiology is discussed.
- Published
- 1984
26. Estrogen receptor status and estradiol sensitivity of MCF-7 cells in exponential growth phase.
- Author
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Madeddu L, Legros N, Devleeschouwer N, Bosman C, Piccart MJ, and LeClercq G
- Subjects
- Cell Count, Cell Line, Electrophoresis, Polyacrylamide Gel, Female, Humans, Neoplasm Proteins analysis, Spectrophotometry, Time Factors, Breast Neoplasms analysis, Estradiol pharmacology, Receptors, Estrogen analysis, Tumor Cells, Cultured drug effects
- Abstract
Proliferative patterns of MCF-7 human breast cancer cells have been reported to influence their estrogen receptor (ER) contents. However, the experimental conditions under which these variations in ER contents were described differed from those commonly used for maintaining exponential growth. We, therefore, investigated whether or not MCF-7 receptor status also fluctuated under normal growth conditions. MCF-7 cells were cultured up to 4 days in 96-multiwell dishes. On each day, cell number was spectrophotometrically assessed after fixation and coloration of the cells with hematoxylin; corresponding ER content was measured by the Abbott enzyme immunoassay in KCl extracts. At the three plating densities tested (5, 10 and 20 x 10(3) cells/ml), an obvious parallel was found between the cell number and the ER content suggesting an unchanged receptor status throughout the culture period. Regression analysis confirmed this impression. Additional fractionation by SDS-PAGE of total MCF-7 proteins extracted at various times of the culture (up to 7 days in 35 mm Petri dishes) gave identical patterns suggesting that ER synthesis is regulated as the majority of proteins. Growth experiments indicated that this situation conferred a constant estrogenic sensitivity to the cells: 24 h exposure to 10(-8) M estradiol on either the 1st, 2nd, 3rd or 4th day after plating resulted in the same increase in cell number. All these data indicated that ER contents of MCF-7 cells were maintained at a constant level under exponential growth which resulted in a constant estrogenic sensitivity.
- Published
- 1988
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27. Membrane fusion in exo-endocytosis: sorting of vesicle and plasma membrane components.
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Madeddu L, Hashimoto S, and Meldolesi J
- Subjects
- Animals, Cell Membrane ultrastructure, Cell Fusion, Endocytosis, Exocytosis
- Published
- 1988
28. Leptinotoxin-h action in synaptosomes and neurosecretory cells: stimulation of neurotransmitter release.
- Author
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Madeddu L, Saito I, Hsiao TH, and Meldolesi J
- Subjects
- Adrenal Gland Neoplasms metabolism, Adrenal Gland Neoplasms ultrastructure, Animals, Calcium metabolism, Dopamine metabolism, Dose-Response Relationship, Drug, Guinea Pigs, Ion Channels drug effects, Microscopy, Electron, Neurosecretory Systems metabolism, Neurosecretory Systems ultrastructure, Pheochromocytoma metabolism, Pheochromocytoma ultrastructure, Synaptosomes metabolism, Synaptosomes ultrastructure, Insect Proteins, Neurosecretory Systems drug effects, Neurotoxins pharmacology, Neurotransmitter Agents metabolism, Proteins pharmacology, Synaptosomes drug effects
- Abstract
Guinea pig brain cortex synaptosomes and neurosecretory PC12 cells were loaded with [3H]3,4-dihydroxyphenylethylamine ([3H]DA, [3H]dopamine) and then exposed to leptinotoxin-h (LPTx) (purified and partially purified preparations, obtained from the hemolymph of Leptinotarsa haldemani). In a Ca2+-containing Ringer medium the toxin induced prompt and massive release of the neurotransmitter. Half-maximal effects were obtained at concentrations estimated of approximately 3 X 10(-11) M for synaptosomes, and 1.5 X 10(-10) M for PC12 cells. Release responses in the two experimental systems investigated were dependent to different extents on the Ca2+ concentration in the medium. In synaptosomes clear, although slow, release of [3H]DA was elicited by the toxin even in Ca2+-free, EGTA-containing medium, provided that high (in the 10(-10) M range) concentrations were used; near-maximal responses were observed at 10(-5)M Ca2+. In contrast, the toxin-induced release from PC12 cells was appreciable only at 3 X 10(-5) M Ca2+, and was maximal at 2 X 10(-4) M and above. In both synaptosomes and PC12 cells Sr2+ and Ba2+ could substitute for Ca2+; Co2+ was inhibitory, whereas Mn2+ failed to modify the release induced by the toxin in Ca2+-containing medium. Organic blockers of the voltage-dependent Ca2+ channel (verapamil and nitrendipine) and calmodulin blocking drugs (trifluoperazine and calmidazolium) failed to inhibit the toxin-induced release of [3H]DA. LPTx induced profound morphological effects. Synaptosomes treated in the Ca2+-containing medium exhibited fusion of synaptic vesicles, formation of numerous infoldings and large cisternae, and alterations of mitochondria. In the Ca2+-free medium the effects were similar, except that their appearance was delayed, and mitochondria were well preserved. Swelling was observed in PC12 cells, accompanied by enlargement of the Golgi area, accumulation of multivesicular bodies, mitochondrial alterations, and decreased number of secretion granules (Ca2+-containing medium). Morphometric analyses revealed a good correlation between the decrease of both synaptic vesicles (synaptosomes) and neurosecretory granules (PC12 cells), and the release of [3H]DA measured biochemically. This is a good indication that the release effect of the toxin is due to stimulation of exocytosis. Taken as a whole, these results confirm the similarity of the effects of LPTx with alpha-latrotoxin of the black widow spider venom, mentioned in the companion article. However, differences in effect and target specificity suggest that the two toxins are specific to separate binding sites.(ABSTRACT TRUNCATED AT 400 WORDS)
- Published
- 1985
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29. "Pure" presynaptic stimulatory toxins and ion transport.
- Author
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Scheer H, Madeddu L, Wanke E, Ferroni A, and Meldolesi J
- Subjects
- Animals, Cell Membrane Permeability drug effects, In Vitro Techniques, Membranes, Artificial, Rats, Receptors, Cholinergic metabolism, Spider Venoms pharmacology, Ion Channels drug effects, Neurotoxins pharmacology, Receptors, Peptide, Synapses drug effects
- Published
- 1985
- Full Text
- View/download PDF
30. Leptinotoxin-h action in synaptosomes, neurosecretory cells, and artificial membranes: stimulation of ion fluxes.
- Author
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Madeddu L, Pozzan T, Robello M, Rolandi R, Hsiao TH, and Meldolesi J
- Subjects
- Adrenal Gland Neoplasms metabolism, Animals, Calcium metabolism, Guinea Pigs, Hemolymph physiology, Insecta, Ion Channels drug effects, Lipid Bilayers metabolism, Membrane Potentials drug effects, Neurosecretory Systems drug effects, Pheochromocytoma metabolism, Proteins isolation & purification, Rats, Insect Proteins, Membranes, Artificial, Neurosecretory Systems cytology, Neurotoxins pharmacology, Proteins pharmacology, Synaptosomes drug effects
- Abstract
Leptinotoxin-h (LPTx), a neurotoxin (otherwise designated beta-leptinotarsin-h) known to stimulate the release of neurotransmitters from synapses, was purified from the hemolymph of the potato beetle, Leptinotarsa haldemani, by a simplification of the procedure originally developed by Crosland et al. [Biochemistry 23, 734-741, (1984)]. Highly and partially purified preparations of the toxin were applied to guinea pig synaptosomes and neurosecretory (PC12) cells. When applied in a Ca2+-containing Ringer medium, at concentrations in the 10(-11) - 10(-10) M range, the toxin induced: (a) rapid depolarization of the plasma membrane, which was not inhibited by organic blockers of voltage-dependent Na+ and Ca2+ channels (tetrodotoxin or verapamil); (b) large 45Ca influx; and (c) increased free cytosolic Ca2+ concentration. These latter two effects were unaffected by verapamil. In Ca2+-free media the effects of the toxin were different in the two systems investigated. In synaptosomes, depolarization was still observed, even if the toxin concentrations needed were higher (approximately 10X) than those effective in the complete medium. In contrast, in PC12 cells no effect of the toxin on membrane potential was observed. Binding of LPTx to its cellular targets could not be investigated directly because the toxin was inactivated by the procedures used for its labeling. Indirect evidence suggested however that Ca2+ is necessary for toxin binding to PC12 cells. Interaction of LPTx with air/water interfaces, as well as with cholesterol/phospholipid mono- and bilayer membranes was investigated. The results indicate that the toxin has affinity for hydrophobic surfaces, but lacks the capacity to insert across membranes unless transpositive voltage is applied. Our results are inconsistent with the previous conclusion of Crosland et al. (1984), who suggested opening of the Ca2+ channel as the mechanism of action of LPTx. The effects of the toxin resemble those of alpha-latrotoxin (alpha-LTx) of the black widow spider venom, and therefore the two toxins might act by similar mechanisms. However, the sites recognized by the two toxins might be different, because LPTx does not inhibit alpha-LTx binding.
- Published
- 1985
- Full Text
- View/download PDF
31. The effect of alpha-latrotoxin on the neurosecretory PC12 cell line: studies on toxin binding and stimulation of transmitter release.
- Author
-
Meldolesi J, Madeddu L, Torda M, Gatti G, and Niutta E
- Subjects
- Animals, Cations, Divalent pharmacology, Cell Line, Clone Cells, Neurosecretory Systems cytology, Spider Venoms metabolism, Arthropod Venoms pharmacology, Dopamine metabolism, Neurosecretory Systems drug effects, Norepinephrine metabolism, Spider Venoms pharmacology
- Abstract
alpha-Latrotoxin of black widow spider venom was found to bind with high affinity (KA = 1.8 X 10(9)M-1) to specific sites present in discrete number (approximately 6300/cell, approximately 12/micron2) at the surface membrane of PC12 cells. This binding correlated with (and therefore, probably caused) the secretory response produced by the toxin. Binding was enhanced (approximately 2-fold) in the presence of mM concentrations of various divalent cations (Ca2+, Mn2+ and Co2+) while Ba2+ and Sr2+ had a smaller effect and Mg2+ was inactive. Hypertonicity, concanavalin A and trypsin pretreatment of the cells blocked the binding interaction. The alpha-latrotoxin-induced stimulation of 3H-dopamine release was massive and occurred very rapidly when cells were exposed to the toxin in a Ca2+-containing Krebs-Ringer medium, whereas it occurred at a much slower rate in a Ca2+-free, Mg2+-containing Ringer. Introduction of Ca2+ into the latter medium resulted in a shift of the release rate from slow to fast. In contrast, in divalent cation-free medium the response was abolished. The toxin-induced secretory response was unaffected by Na+ and Ca2+ channel blockers (tetrodotoxin and D600) as well as by calmodulin inhibitors (calmidazolium and trifluoperazine). The effects of Ca2+ and Mg2+ were found to be concentration-dependent, with half maximal responses occurring at approximately 0.3 and 1.5 mM for the two divalent cations, respectively. Other divalent cations could substitute for Ca2+ and Mg2+, the relative efficacy being Sr2+ greater than Ca2+ greater than Ba2+ much greater than Mn2+ greater than Mg2+ greater than Co2+. Moreover, the response occurring at suboptimal concentration of Ca2+ (0.4 mM) was potentiated by the concomitant addition of either Mg2+, Mn2+ or Co2+. The effect(s) of divalent cations in supporting the alpha-latrotoxin-induced release response seem(s) to occur primarily at step(s) beyond toxin binding because (a) the stimulatory effects of the various cations on release were not matched by parallel effects on binding, and (b) Ca2+ maintained its ability to stimulate fast release even when toxin binding had occurred in a Ca2+-free medium. Delays in the release responses were observed when cells were exposed to alpha LTx in Na+-free, glucosamine or methylamine-based media, or depolarized with high K+ (in the presence of D600) before toxin treatment. Moreover, in these two conditions the ability of Mg2+ to support the alpha LTx response was considerably decreased.(ABSTRACT TRUNCATED AT 400 WORDS)
- Published
- 1983
- Full Text
- View/download PDF
32. A new method for evaluating the inhibitory potency of antiestrogens on breast cancer cell lines.
- Author
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Madeddu L, Roy F, and Leclercq G
- Subjects
- Cell Count, Cell Division drug effects, Cell Line, Estradiol pharmacology, Female, Humans, Methods, Nafoxidine therapeutic use, Nitromifene therapeutic use, Spectrophotometry, Staining and Labeling, Tamoxifen analogs & derivatives, Tamoxifen therapeutic use, Tetrahydronaphthalenes therapeutic use, Breast Neoplasms drug therapy, Estrogen Antagonists therapeutic use
- Abstract
Breast cancer cell lines in monolayer culture are helpful in evaluating the potential antitumor activity of antiestrogens. A new method has been developed to rapidly measure the inhibitory potency of a given compound without any cell harvest and counting. Cells are incubated in 96-multiwell culture dishes in the absence or presence of an antiestrogen at 3 concentrations. At the end of incubation, cells are fixed with ethanol and colored with hematoxylin. The intensity of the coloration, which is measured with a multiscan spectrophotometer, gives an accurate evaluation of the cell number and, thereby, an estimate of the inhibitory potency of the antiestrogen. Addition of estradiol to the culture allows to evaluate the antagonistic effect of this hormone towards the inhibition of the antiestrogen. Because of its rapidity and low cost, our method should offer a new dimension in the in vitro screening of antiestrogens and probably most other antineoplastic drugs.
- Published
- 1986
33. Intracellular calcium homeostasis in a human neuroblastoma cell line: modulation by depolarization, cholinergic receptors, and alpha-latrotoxin.
- Author
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Sher E, Gotti C, Pandiella A, Madeddu L, and Clementi F
- Subjects
- Aminoquinolines, Atropine pharmacology, Calcimycin pharmacology, Carbachol pharmacology, Cytoplasm metabolism, Egtazic Acid pharmacology, Fluorescent Dyes, Humans, Ion Channels drug effects, Ion Channels physiology, Membrane Potentials, Pirenzepine pharmacology, Potassium Chloride pharmacology, Receptors, Muscarinic drug effects, Receptors, Muscarinic physiology, Receptors, Nicotinic drug effects, Receptors, Nicotinic physiology, Spectrometry, Fluorescence, Tumor Cells, Cultured, Verapamil pharmacology, Arthropod Venoms pharmacology, Calcium metabolism, Homeostasis, Neuroblastoma metabolism, Receptors, Cholinergic physiology, Spider Venoms pharmacology
- Abstract
Intracellular calcium homeostasis and its modulation by different agents was studied in control and differentiated IMR32 human neuroblastoma cells by using the Ca2+-sensitive fluorescent dye quin2. The results obtained demonstrate the existence in IMR32 cells of (a) voltage-dependent, verapamil sensitive, Ca2+ channels, which are expressed before differentiation; (b) muscarinic receptors whose activation triggers both Ca2+ influx and Ca2+ redistribution from intracellular stores, whereas nicotinic receptors and alpha-bungarotoxin binding sites do not; and (c) receptors for alpha-latrotoxin (the major toxin of the black widow spider venom), which are well-known markers of the neuronal presynaptic membrane. Up to now, no cell lines of human origin sensitive to this toxin have been identified. These results confirm that IMR32 cells are very convenient model cells for studying specific aspects of the neurochemistry and neurobiology of the human neuron at the molecular and cellular levels.
- Published
- 1988
- Full Text
- View/download PDF
34. Black widow spider venom-induced release of neurotransmitters: mammalian synaptosomes are stimulated by a unique venom component (alpha-latrotoxin), insect synaptosomes by multiple components.
- Author
-
Knipper M, Madeddu L, Breer H, and Meldolesi J
- Subjects
- Acetylcholine metabolism, Animals, Corpus Striatum drug effects, Corpus Striatum metabolism, Dopamine metabolism, Electrophoresis, Polyacrylamide Gel, Ganglia drug effects, Ganglia metabolism, Grasshoppers, Rats, Rats, Inbred Strains, Spider Venoms analysis, Synaptosomes metabolism, Arthropod Venoms pharmacology, Neurotransmitter Agents metabolism, Spider Venoms pharmacology, Synaptosomes drug effects
- Abstract
Synaptosomes isolated from the rat brain corpus striatum and locust head and thoracic ganglia were loaded with radioactive neurotransmitter ([3H]dopamine and [3H]acetylcholine, respectively) and then treated with alpha-latrotoxin and other fractions (fractions C, D and E of Frontali et al.8) obtained by Sephadex G200 column chromatography from black widow spider venom gland homogenates. As shown by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, alpha-latrotoxin is a high Mr protein, whereas fractions C-E are mixtures of several proteins, that include small amounts of contaminating alpha-latrotoxin (especially in fraction C). In rat synaptosomes alpha-latrotoxin induced massive neurotransmitter release, and some release was induced also by high concentrations of fractions C and D. These responses were blocked almost completely by a monospecific anti-alpha-latrotoxin serum, indicating that they were all due to alpha-latrotoxin. Release of [3H]acetylcholine from locust synaptosomes was induced by the various preparations investigated. alpha-Latrotoxin was about 10-fold less potent in locust than in rat synaptosomes. The effects of fractions C-E tended to disappear with storage. The most active batches of fractions C and E were even more potent than alpha-latrotoxin, while the D fraction was approximately 5-fold less potent. The anti-alpha-latrotoxin antiserum inhibited part of the responses elicited by fractions C and E, but left fraction D almost unaffected. Release by D and E fractions was maintained even when Ca2+ was removed from the incubation medium.(ABSTRACT TRUNCATED AT 250 WORDS)
- Published
- 1986
- Full Text
- View/download PDF
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