Showing papers by "University of Buenos Aires published in 1971"

Angiotensin and renin in rat and dog brain.

Abstract: An enzyme with the characteristics of classical renin was isolated from brain extracts of nephrectomized dogs. This enzyme is thermolabile, nondialyzable, and forms a vasoconstrictive material when incubated with renin plasma substrate at pH 7. A short lasting pressor activity was also found in brain extracts of dogs and rats. This activity was due to a substance identified by chemical and pharmacological tests as angiotensin. Countercurrent distribution of brain extracts of rats showed that 38% of the pressor activity corresponded to angiotensin II and the remainder to angiotensin I. A remarkable correlation was found between angiotensin and norepinphrine concentrations in different portions of the encephalon of the dog.

Molecular Biology of Synaptic Receptors

Abstract: A special proteolipid (a hydrophobic protein) has been extracted and purified from nerve-ending membranes and total particulate matter of gray areas of the central nervous system. Such a proteolipid shows a high affinity for binding d-tubocurarine, serotonin, and atropine and has been called receptor proteolipid. The interaction of this proteolipid with atropine sulfate was studied with light scattering and polarization of fluorescence. The changes observed, which follow a cooperative type of curve, were attributed to the aggregation of the proteolipid macromolecules. Such a phenomenon was then observed under the electron microscope. A receptor proteolipid having a high affinity for binding acetylcholine, hexamethonium, and other cholinergic drugs was isolated and purified from electric tissue of fishes and from electroplax membranes. Such a proteolipid was also extracted from membranes from which acetylcholinesterase had been removed, and it was concluded that this enzyme and the receptor proteolipid are two different macromolecules. A high affinity binding site with a dissociation constant of K1 equal to 10 -7 and about ten sites with K2 equal to 10 -5 were recognized in the receptor proteolipid. Under the electron microscope the receptor proteolipid of brain appears as a rod-shaped macromolecule which may assume paracrystalline arrays with 10 -8 molar atropine sulfate. Similarly the receptor proteolipid from electric tissue and from skeletal muscle may form paracrystalline arrays under the action of acetylcholine and hexamethonium. A model of the cholinergic receptor based on the properties of the proteolipid is presented. Preliminary work indicates the possibility of obtaining a biophysical response to acetylcholine when the receptor proteolipid is embedded in artificial bilayered lipid membrance.

Independence of protein synthesis and drug uptake in nerve cell bodies and glial cells isolated by a new technique.

Abstract: A severe handicap in any study of the cellular biochemistry of the brain is the unavailability of sufficient amounts of pure neurones and glial cells. Many efforts to separate one from the other have been made1, but only four basic types of isolation procedures have been described for routine use: (1) brain is treated with a mixture of acetone-glycerol and water before mincing and centrifugation3–5; (2) brain mince is sieved and subjected to gradient centrifugation1,6–8—this separates neuronal cell bodies from neuropil6 or from intact but rather contaminated glial cells8; (3) whole brain is disrupted in a tissue press and zonal centrifugation is used to separate cellular and subcellular components as they leave the zonal centrifuge rotor9; and (4) chopped brain is incubated in oxygen at 37° C in the presence of 1 % (w/v) trypsin10, a treatment which, in our estimation, severely prejudices this technique's general usefulness, because it precludes meaningful comparative investigations of enzymes and non-enzymatic structural and/or soluble cell proteins. Since investigations of the latter type are the aim in our laboratory, we needed a technique in which brain is not subjected to deleterious treatments such as immersion in acetone-glycerol-water mixtures3–5 or trypsinization10 and in which no special instrumentation9, tissue presses9 or homogenizers8 are required. We now report a technique which successfully accomplishes this objective, for it makes possible the isolation of highly purified neuronal perikarya and of intact glial cells from a few grams of brain, in yields which permit the subsequent subcellular fractionation of the isolated cells. We also report two applications of the new technique which have enabled us to determine the in vivo time course of protein synthesis in, and the uptake of the radioactive convulsant agent methionine sulphoximine11,12 by, the neurones and the glial cells of the immature rat brain cortex.

DNA-Protein Complex in Circular DNA from Phage ϕ29

Abstract: THE DNA of the B. subtilis phage ϕ29 has been described as unpermuted linear duplex molecules1 of molecular weight 11 × 106, but the formation of circular molecules has also been indicated, suggesting the existence of cohesive ends1,2.

Regulation of the Nucleolar DNA-Dependent RNA Polymerase by Amino Acids in Ehrlich Ascites Tumor Cells

Abstract: Experiments were performed to ascertain the degree to which the amount of amino acids might be one of the regulatory factors that control the activity of the nucleolar RNA polymerase. Assays of the enzymatic activity were done with isolated nuclei from cells incubated with low and high concentrations of amino acids. Soon after the cells were exposed to a medium enriched in amino acids, a rapid increase of nucleolar RNA polymerase activity occurred. A similar result was obtained in cells incubated with lower concentrations of amino acids. However, the rate of ribosomal RNA synthesized was regularly much higher in cells incubated in a medium enriched with amino acids than in a medium low in amino acids. Apparently, the amino acids only controlled ribosomal RNA synthesis. Thus, neither maturation, processing, and transport of nuclear precursors into cytoplasmic ribosomal RNA, nor the synthesis of rapidly labeled RNA was affected.

Conductance Changes Produced by Acetylcholine in Lipidic Membranes Containing a Proteolipid from Electrophorus

Abstract: Ultrathin lipidic membranes containing one ten-thousandth of a special proteolipid from electric organ of Electrophorus reacted to the addition of acetylcholine by a rapid and transient increase in conductance. Such a change was not induced by choline and is greatly reduced by a previous application of d-tubocurarine. These properties, resembling those from chemically excitable membranes, were not observed with another proteolipid from the same tissue.

The effect of immuno-depression due to neonatal thymectomy on infections with trypanosoma cruzi in mice

Abstract: Neonatally thymectomized C3H or Rockland mice infected with trypanomastigotes of Trypanosoma cruzi had higher parasitaemia and shorter survival than their non-thymectomized counterparts. The appearance of antibodies detectable by IFT, IHA or DAT was delayed in neonatally thymectomized mice infected with epimastigotes of T. cruzi. Less thymectomized than non-thymectomized mice survived infection with epimastigotes. Our findings suggest that thymus-dependent, immune mechanisms are involved in controlling infection with T. cruzi in mice.

Multiple binding sites for acetylcholine in a proteolipid from electric tissue.

Abstract: A special proteolipid protein fraction having a high affinity for binding to acetylcholine and other cholinergic drugs was isolated from electroplax of Electrophorus electricus . After extraction in chloroform-methanol (2:1 by volume) and column chromatography on Sephadex LH-20, the proteolipid appeared in a special peak together with the radioactive acetylcholine and increased in proportion with the amount of protein applied to the column. A saturation curve of this peak with respect to concentrations of 14C-acetylcholine between 7 x 10-7 and 5 x 10-5 M was constructed. The shape of this curve and the double-reciprocal plot of the data suggested the presence of more than one type of binding site. Assuming a homogeneous population of molecules having a molecular weight of 40,000, the use of the Scatchard equation suggested that there is a single high-affinity binding site with an apparent dissociation constant of 1 x 10-7 M and a group of low-affinity sites with a dissociation constant of 1 x 10-5 M. Since this proteolipid is present only in the electroplax membranes, these results are discussed in relation to the possible quantity of this material per electroplax or synaptic junction and its possible physiological significance as a receptor in cholinergic synapses. ACKNOWLEDGMENTS The authors are indebted to Professor Gregorio Weber of the University of Illinois, to Dr. Enrique Rosengurt of the Instituto de Investigaciones Bioquimicas, and Dr. George G. Lunt for their helpful suggestions in the analysis of the data.

[Increased incorporation of [G-3H]leucine into a possible "receptor" proteolipid in denervated muscle in vivo.

Abstract: — The incorporation of radioactive leucine into the total proteins and the proteolipids of normal and denervated rat diaphragm has been studied in vivo. Denervation increased the incorporation of isotopically labelled leucine into each of the isolated proteolipids and the effect was particularly marked in a single proteolipid which has been designated a ‘receptor’ proteolipid. In normal muscle this particular proteolipid was found to have a higher incorporation of isotopically labelled leucine in the area of the muscle rich in endplates compared with an area devoid of endplates. However the stimulatory effect of denervation on the incorporation of radioactive leucine into this proteolipid was considerably more marked in the latter region. An attempt has been made to correlate these findings with the development of the hypersensitivity to ACh characteristic of denervated muscle.

Histometric evaluation of periodontal bone loss in rats. I. The effect of marginal irritation, systemic irradiation and trauma from occlusion.

Abstract: The effect of marginal irritation, trauma from occlusion and total body X-irradiation on interradicular bone of Wistar rat molars, was studied after an experimental period of two weeks. Histologic findings and slatistical evaluation of histometric results indicate that: – the only single factor capable of reducing the height of interradicular bone was marginal irritation, – the only additional factor that was capable of increasing this reduction in height was total body X-irradiation, – marginal irritation, either alone or in combination with trauma from occlusion increased bone density in the submarginal areat – total body X-irradiation inhibited the osteogenic reaction induced in the submarginal area by marginal irritation. – trauma from occlusion did not have any effect on the parameters measured.

2-Amine 4-Pentenoic Acid (Allylglycine): A Proposed Tool For The Study of Gaba Mediated Systems

Abstract: Structural and biochemical alterations produced by the convulsant drug 2-amine-4-pentenoic acid (allylglycine) in the cerebellar cortex are described. Most Purkinje cells show considerable shrinkage, condensation and darkening of the cytoplasm and nucleus and the basket synapses appear swollen and with loss of synaptic vesicles. This contrasts with the normal aspect of granular cells and glomerular synapses. In the cerebellum of the convulsant rat there is 32–35 % inhibition of glutamic acid decarboxylase. Kinetic studies suggest that the inhibition is due to a competitive inhibition for the substrate. These results suggest that allylglycine may be used as a tool for the study in the central nervous system of inhibitory systems mediated by γ-aminobutyric acid.

Maintenance of Nitrogen Balance in a Young Woman by Substitution of α-Ketoisovaleric Acid for Valine

Abstract: Negative nitrogen balance was induced in a young female by removing valine from a diet containing 10.3 g of nitrogen composed of glycine, diammonium citrate and the eight essential L-amino acids in the proportion present in egg protein. Addition of a-ketoisovaleric acid decreased negative nitro gen balance and reverted it to equilibrium when an amount equivalent to three times that of valine was administered. Negative nitrogen balance returned when o-keto acid supplement was withdrawn. J. Nutr. 101: 1165-1168, 1971.

Role of the pia mater in the transfer of substances in and out of the cerebrospinal fluid

Abstract: The transfer of 14C leucine, 14C GABA and 14C urea from an isolated CSF compartment has been studied by implanting plastic cups on the cerebral cortex of anaesthetized cats under different experimental conditions. Removal of the pia mater resulted in a significant decrease in the disappearance of the substances from the cups. The intravenous infusion of GABA also stopped the escape of this amino acid from the plastic cups. When the cups contained only artificial CSF, and GABA was infused intravenously, its appearance in the cups followed a diffusional pattern. Ligaturing the sagittal venous sinus decreased the rate of disappearance of 14C urea from the plastic cups. Higher concentrations of GABA and leucine were found in the pial membrane than in the underlying brain tissue, after these amino acids in solution were left in the cortical cups for 5 hours. Pressure applied during implantation of the cups sometimes caused accumulation of fluid in the cups. It is suggested that the pial membrane and its vasculature play an important role in the transfer of substances in and out of the subarachnoid space.

Association of the Acetylcholine-phosphatidyl Inositol Effect with a “Receptor” Proteolipid from Cerebral Cortex

Abstract: A striking effect of acetylcholine is the stimulation of phosphatidyl inositol turnover in nervous1–3 and other tissues1,4 as witnessed by the increased incorporation of 32P and isotopically labelled inositol. The effect has been observed in vitro in brain slices1,2 and in brain homogenates5–7 and has been localized in subcellular fractions rich in nerve endings8. More recently a marked increase in the 32P incorporation into phosphatidyl inositol, but not into other phospholipids, was demonstrated in electrically stimulated slices of cerebral cortex9. Preganglionic stimulation of sympathetic ganglia also resulted in an increased phosphatidyl inositol turnover and indirect evidence was presented that the effect occurred at the postsynaptic membrane10,11. These and other findings imply that phosphatidyl inositol may play a special part in synaptic transmission but the mechanisms involved in the acetylcholine effect are as yet unsolved.

Evidence for a Normal Geomagnetic Field Polarity Event at 263 ± 5 m.y. BP within the Late Palaeozoic Reversed Interval

Abstract: Basalts, radiometrically dated at 263 ± 5 my, have stable remanent magnetization with normal polarity This demonstrates the existence of a previously unrecognized event within the Late Palaeozoic Interval of reversed geomagnetic polarity

Fine structure of ultrathin artificial membranes : I. Changes by acetylcholine addition in lipid proteolipid membranes.

Abstract: A special technique for the electron-microscope study of the fine structure of ultrathin artificial membranes is described. Membranes made of total phospholipids of the cerebral cortex and cholesterol showed globular elements of 40 A embedded in a denser and diffuse matrix. These same elements were also seen organized in a periodic banded pattern. Identical patterns were observed with and without supporting films. Lipidic membranes containing small amounts of proteolipid fromElectrophorus showed a lower electron density, a finer and smoother texture and a decrease in electrical resistance.Lipidic membranes containing the cholinergic receptor proteolipid fromElectrophorus, upon addition of acetylcholine, showed a rapid and transient rise in conductance which was accompanied by changes in fine structure, consisting in a more uneven corrugated appearance of the membrane and the presence of dense spots of 20 A. These results are discussed in relation to the channel hypothesis of ion permeability. It is postulated that the binding of acetylcholine by the receptor proteolipid results in conformational changes in this protein that facilitate the translocation of ions through the membrane.

Carotid body catecholamines. Histochemical studies on the effects of drug treatments.

Abstract: Histochemical studies were made in the cat's carotid body using two fluorescence techniques: the formaldehyde condensation (HCOH) method and the trihydroxyindole (THI) method. A number of pharmacologic agents known to alter catecholamine metabolism or binding were used to evaluate their effect on the amines found in the glomus cells. After treatment with reserpine, both histochemical techniques showed a reduction and eventual disappearance of fluorescence from the glomus cells. Treatment with a monoamine oxidase inhibitor (iproniazid) or with dopamine beta hydroxylase inhibitor (disulfiran) enhanced the glomus cell fluorescence. The observed increase was greater with the THI than with the HCOH technique. A few yellow fluorescent cells were found following a combination of reserpine and iproniazid treatments. A reduction in fluorescence with both techniques was obtained following DOPA decarboxylase inhibitors (MK-485). It is concluded that some of the glomus cells contain only dopamine while others contain norepinephrine or a combination of norepinephrine and dopamine. In addition the presence of DOPA in some cells following treatment with pharmacologic agents may account for some of the results. Finally, the few yellow fluorescent cells found probably contain 5-hydroxytryptamine.