Light and electron microscopy of granules in the toad choroid plexus

Summary Two types of granules can be distinguished in the toad choroid plexus under the light microscope: pigment granules, mainly localized in the cells that line the free ends of the choroidal villi, and Gomori-positive granules, present in most epithelial cells.The ultrastructural analysis of the choroid plexus reveals three types of granules: multivesicular bodies (MVB), multigranulous bodies (MGB) and dense bodies (DB), and intermediate stages between the last two bodies. The pigment granules seen under the light microscope probably correspond to the DB of the electron micrographs, and the Gomori-positive granules to the MGB. The probable role of these bodies is discussed and so is the significance of the glycogen present in the choroidal cells, their processes and endothelium.This study was partially supported by the Consejo Nacional de Investigaciones Científicas y Técnicas de la República Argentina and the Rockefeller Foundation (School grant RF — 58028).Fellow of the Consejo Nacional de Investigaciones Científicas y Técnicas de la República Argentina. The author wishes to thank Prof. M. H. Burgos for his constant interest. His thanks are also due to Prof. H. Heller for providing certain facilities in his department and for his criticism.     

Effect of exogenous application of gibberellic acid on color change and phenylalanine ammonia-lyase, chlorophyllase, and peroxidase activities during ripening of strawberry fruit (Fragaria x ananassa Duch.)

The effect of exogenously applied gibberellic acid (GA₃) on the postharvest color change of strawberry fruit was evaluated through their external color and surface color parameters. A significant delay on color evolution was observed in fruits treated with GA₃. The evolution of activities of phenylalanine ammonia-lyase (PAL), chlorophyllase, and peroxidase was also analyzed. PAL activity increased during strawberry ripening, but in fruits treated with GA₃ the increase in such activity was slower, and, probably as consequence, the development of red color was delayed. Moreover, the activity of chlorophyllase and peroxidase, enzymes possibly involved in chlorophyll metabolism, decreased during strawberry ripening. However, a delay was observed in the decrease of such activities in GA₃-treated fruits.Abbreviations PAL phenylalanine ammonia-lyase - GA₃ gibberellic acid₃ - PVPP polyvinylpolyprrolidone - CEAU chlorophyllase enzymic activity unit - PEAU peroxidase enzymic activity unit - LSD least significant difference. Fellow of the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) de la República Argentina. Author for correspondence.Members of the Research Career of the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) de la República Argentina.     

Vascularization of the hypophysial region of the normal and adenohypophysectomized toad

Summary The study of standarized sections of the hypophysial regions, and in vivo observations showed the presence of communicating vessels between the capillary network of the median eminence and the large capillaries of the neurointermediate junction. Moreover, direct branches from the hypophysial artery are described which give off branches, at the level of the neural stalk, to the median eminence and to the large capillaries of the neurointermediate junction.A second portal system similar to the one described by Cruz has been observed. Its primary plexus originates in several encephalic regions, and its secondary plexus is distributed through the neural lobe and thence to the pars intermedia. The course of flow in this system is a descending one. The arterial contribution to this system appears to arise from branches from the basilar and retroinfundibular arteries. — There are small venous-type vessels between the large capillaries of the neurointermediate junction and the posterodorsal region of the pars distalis. — After adenohypophysectomy, the blood which normally goes towards the pars distalis, flows towards the pars intermedia, following the path of the communicating vessels between the median eminence and the pars intermedia.This paper was presented at the VII Reunion de la Asociación Latinoamericana de Ciencias Fisiológicas (A.L.A.C.F.), Mar del Plata, Argentina, 1966. It was carried out under the auspices of the Consejo Nacional de Investigaciones Científicas y Técnicas de la República Argentina and the Rockefeller Foundation (School grant RF-58028).Fellows of the Consejo Nacional de Investigaciones Científicas y Técnicas de la República Argentina. — The authors wish to thank Prof. B. A. Houssay and Drs. J. H. Tramezzani and J. la Pointe for their criticism, to Prof. M. H. Burgos and Dr. F. Sacerdote for their help.     

On the presence of conspicuous electron dense bodies in the pinealocytes of the pig

Summary The pinealocytes of the pig contain a large number of aldehydefuchsin positive granules. In order to determine their nature an ultrastructural study was carried out. Numerous bodies having a maximal diameter of about 1,600 nm were found. These elements showed a great variety of internal structure, ranging from a homogeneous content and amorphous dense aggregates to lamellate bodies. Although only a few of them displayed a positive reaction for acid phosphatase, their morphological appearance strongly suggests that they belong to the lysosomal system.Aided in part by a grant from the Consejo Nacional de Investigaciones Cientificas y Técnicas de la República ArgentinaAssociate Professor of Embryology and Histology and Member of the Investigator Career of the CONICETProfessor and Chairman of Histology and Member of the Investigator Career of the CONICETCenter sponsored by the Universidad Nacional de La Plata and the Comisión de Investigaciones Cientificas de la Provincia de Buenos Aires     

Diabetes-induced alteration in subcellular distribution of poly(A)-rich RNA from skeletal muscle

Summary Skeletal muscle ribosome preparations from diabetic rats have lower polysome content than those from normal animals. The ratio of poly(A) containing RNA between polysomes and postribosomal supernatant has been measured both in normal and diabetic rats. The results suggest that in diabetic animals there is a greater proportion of free poly(A) containing RNA, in postribosomal supernatant.This work was supported by Grants from the Consejo Nacional de Investigaciones Cientificas y Técnicas (República Argentina), Universidad Nacional de Buenos Aires, and partly under contract of the Ministére de la Politique Scientifique within the framework of the Association Euratom—University of Brussels—University of Pisa.     

Twenty-four-hour rhythm in the mitotic activity and in the water and dry matter content of regenerating liver

Summary A twenty-four hour rhythm is described in the variations of mitotic activity, dry weight, and water content of the regenerating liver of mice hepatectomized about noon. These variables have their maximal value at the onset of the rest periods in the morning and their minimal values at the initiation of the body activity periods in the evening. The rhythm is well defined on the second day and is repeated on the third day on a higher level.We acknowledge the assistance of Mrs. R. Cavoura in performing the hepatectomies and that of Mrs. M. A. V. Albornoz in making the histologic slides.The present work was carried out with the help of grants from the Consejo Nacional de Investigaciones Científicas y Técnicas de la República Argentina and Liga Mendocina de Lucha contra el Cáncer.     

Glycogen in the neurohypophysis of toads

Summary Studies with the electron microscope revealed the presence of glycogen granules in the neurohypophysis of toads (Bufo arenarum Hensel). The glycogen appeared as scattered particles or as clusters formed by these particles within the nerve fibers. The scattered granules were intermingled with the neurosecretory granules and with the microvesicles. Chemical assays showed the presence of glycogen in the neurointermediate lobe of the toad's hypophysis.This work was supported by research grants from the Consejo Nacional de Investigaciones Científicas y Técnicas de la República Argentina and The Rockefeller Foundation.The authors wish to express their appreciation to Prof. Bernardo A. Houssay and to Dr. Luis F. Leloir for their encouragement during the preparation of this paper; to Miss Susana Camocardi for her technical assistance and to Mr. Luis Millara for the photographic work.     

Use of a packed-bed reactor for anaerobic treatment of stillage of sugar cane molasses

Summary The use of a packed-bed reactor for the treatment of stillage from sugar cane molasses is studied in a laboratory scale unit of 250 ml at 40°C. When stability is reached organic loads from 4 to 30 g COD/l/day are applied with COD reduction ranging from 85% to 55% depending on the load and the source of stillage. The production of biogas achieved is of 379 1 (72% CH₄) per Kg COD removed.Career investigator of the Consejo Nacional de Investigaciones Científicas y Técnicas (Argentina).Fellow of the Consejo Nacional de Investigaciones Cientifícas y Técnicas (Argentina).     

Ependymal specializations

Summary The primary plexus of the toad hypothalamic-adenohypophysial portal system has two types of loops. The short loops are localized in the external region of the median eminence and surrounded by nerve endings and glial cells. The long loops approach the ependymal lining of the median eminence. The ascending and descending branches of these loops are surrounded by nerve and ependymal endings and glial cells. The actual subependymal portion of the long loops is virtually in contact with ependymal processes only, which form a cuff interposed between this portion of the long loops and the fibres of the hypothalamic-neurohypophysial tract. Many of the vascular endings of the ependymal processes have electron dense granules whose diameter ranges between 700 and 1400 Å. The ultrastructure of the ependymal cells suggests that these granules are transport material and not secretory material.This anatomical arrangement linking the ependyma of the median eminence and the long loops of the primary plexus of the hypothalamic-adenohypophysial portal system makes the possibility of an interrelationship between the cerebrospinal fluid and the portal blood very considerable.Fellow of the Consejo Nacional de Investigaciones Científicas y Técnicas de la República Argentina. The author takes great pleasure in thanking Prof. H. Heller for his constant interest and criticism.     

Regeneration of plants from callus tissue of the pasture legume Lotononis bainesii

In vitro regeneration of plants from both cotyledon-, and leaf — derived calli of Lotononis bainesii Paker was studied under defined nutritional, hormonal and environmental conditions. Explants from both, cotyledons from seedilings of 4 days old and fully expanded leaves from mature plants, were cultured on MS medium containing 0.8% agar and supplemented with 0.01, 0.1, and 1 mg/1 concentrations of naphthaleneacetic acid (NAA) and 0.1, 1, and 3 mg/1 levels of benzyladenine (BA) in various combinations. Multiple shoot (on an average 4 shoots per callus) regeneration from primary callus occurred within 15 to 35 days of culture in most of the media tested. Although the best medium for shoot regeneration from cotyledon-derived callus contained NAA and BA at 1, and 0.1 mg/1 levels, respectively, maximal shoot regeneration from leaf-derived calli was achieved by using NAA and BA at 0.01 and 0.1 mg/1, respectively. Roots were induced to differentiate by transferring the regenerated shoots onto a medium lacking growth regulators.Supported by a research fellowship from Consejo Nacional de Investigaciones Científicas y Técnicas (República Argentina).     

Differences between median eminence and neural lobe in amphibians

Summary The neurohypophysis of the toad Bufo arenarum Hensel can be subdivided into two well defined zones: the median eminence and the neural lobe. In its turn, the median eminence consists of two zones: a neural subependymal one, containing the axons of the hypothalamic-neurohypophysial tract; and a glandular one, made up of the capillaries of the primary plexus of the portal hypophysial system and of neurosecretory axons.Different staining techniques and certain experimental procedures (inanition, dehydration and adenohypophysectomy) showed that there are differences between the neurosecretory material of the neural lobe, which is a place of hormone liberation into the systematic circulation, and the neurosecretory material of the median eminence, which is the site of release of adenohypophysis-stimulating substances into the portal vessels.This work was introduced at the III Sesiones Científicas de Biología, Rosario, Argentina, March, 1965. The study was partially supported by the Consejo Nacional de Investigaciones Científicas y Técnicas (Argentina) and The Rockefeller Foundation (School grant RF-58028).Fellow of the Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina. The author wishes to express his indebtedness to Dr. M. H. Burgos and Dr. F. Sacerdote for their help and criticism, to Miss B. Rodriguez. Miss M. Masot and Mr. L. Castro for their technical assistance.     

Cytochemistry of 5-hydroxytryptamine at the electron microscope level

Summary The nerves of rat pineal gland are known to contain norepinephrine and 5-hydroxytryptamine. With the glutaraldehyde-dichromate reaction for the cytochemical localization of unsubstituted catechol- and indoleamines, dense reactive granules could be demonstrated in such endings. A similar reaction was observed in the adrenergic nerves supplying the vas deferens and storing exclusively norepinephrine. Formaldehyde fixation, prior to the glutaraldehyde-dichromate treatment, interferes with the reaction given by catecholamines not affecting the indolic reactive sites. After this combined procedure pineal nerves still exhibited the dense reactive granules, while these were not found in the nerves of the vas deferens. Following bilateral cervical sympathectomy reactive granules disappeared from the perivascular processes of the pineal gland. No reaction could be observed in the cytoplasm of parenchymal cells neither in their perivascular processes.These cytochemical results suggest that both catecholamines and 5-hydroxytryptamine are contained within the granulated vesicles of pineal nerves.This work has been supported by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina and U.S.Air Force (AF-AFOSR 963-66).Fellow of the Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina. We want to express our gratitude to Prof. E. De Robertis for his constant help and encouragement; and to Miss Nélida Fernández Oranges, Mr. Raúl Castelli and Mr. Alberto Sáenz for their skillful technical assistance.     

Treatment of dairy industry wastewater with an anaerobic filter

Summary Concentrations of up to 10.2 g COD/L were applied to an horizontal anaerobic filter at 40°C, obtaining efficiencies in COD removal of 85%. The contents of the reactor are kept mixed by recycling and at a pH value of 6.9.The addition of alkali to the influent increases the production of biogas reaching a maximum of 250 L of methane per kg of COD removed.Career Investigator of the Consejo Nacional de Investigaciones Cientificas y Técnicas (R. Argentina).Fellow of the Consejo Nacional de Investigaciones Cientificas y Técnicas (R. Argentina).     

ZIO staining in synaptic vesicles of the rat pineal nerves after inhibition of serotonin and noradrenaline synthesizing enzymes

Summary Two compartments have been defined in monoaminergic synaptic vesicles: the core or central compartment, storage site for monoamines, and the matrix or outer compartment, of unknown function. The outer compartment reacts with the mixture of zinc iodide-osmium tetroxide (ZIO). This reaction is temperature and time dependent and may be abolished by -SH reagents.The effect of drugs inhibiting the synthesis of serotonin and noradrenaline (stored in the core) on the ZIO reaction in the matrix was studied in synaptic vesicles of rat pineal nerves. The inhibitors of monoamine synthesis abolish or decrease the ZIO reaction directly or in combination with the administration of tyramine. This effect is temperature dependent suggesting that the drugs act on different components of the matrix that react with ZIO at different temperatures. A comparison of the present results with those obtained with -SH reagents seems to indicate that the drugs assayed act, at least in part, by changing the accessibility of-SH groups in vesicle proteins. (An abstract of this paper was presented at the 7th International Congress of Pharmacology, Paris, 1978.)Supported by grants from the Consejo Nacional de Investigaciones Cientificas y Técnicas, CONICET, and the Secretaría de Ciencia y Técnica, SECYT, ArgentinaWe are grateful to Margarita López de Cáceres for technical assistance, to Adriana Contreras for the electron micrographs and to María Aued de Rau for the illustrations. Rita Cardoni is a fellow of the Consejo Nacional de Investigaciones Cientificas y Técnicas, Argentina     

Ependymal specializations

Summary The ependymal cells of the toad subcommissural organ produce pale and dense secretory granules. Both types of granules are mainly concentrated in the apical cytoplasm and in the perinuclear region. Pale and dense granules are synthesized by and packed in the rough endoplasmic reticulum, bypassing the step of the Golgi apparatus. The apical cytoplasm of some subcommissural ependymal cells protrudes into the ventricle. All the cells project a few cilia and numerous slender, long microvilli into the ventricular lumen.Contacting the cilia and the microvilli there is a filamentous material identical to that observed in the fibre of Reissner at the aqueduct of Sylvius. In addition to filaments, the fibre of Reissner contains vacuolar formations. The fibre is surrounded by numerous ependymal cilia, some of which are embedded in the filamentous material of the fibre.The presence of numerous microvilli projected into the ventricle and the large number of vesicles scattered in the supranuclear cytoplasm seem to indicate that the subcommissural organ may have absorption functions. The fact that the intercellular space of the ependymal layer of the subcommissural organ is not separated from the ventricular lumen by tight junctions but by zonulae adhaerentes could indicate that the cerebrospinal fluid penetrates these intercellular spaces bathing all sides of the ependymal cells. The presence in the ependymal cells of vesicles opening into the intercellular space would be in agreement with the latter possibility.There are some ultrastructural differences between the ependymal cells of the cephalic end of the subcommissural organ and those of the caudal end. A critical analysis of Reissner's fibre formation is made.This investigation was partially supported by a Grant of the Wellcome Trust Foundation.Fellow of the Consejo Nacional de Investigaciones Científicas y Técnicas de la República Argentina. The author wishes to thank the valuable help of Mr. P. Heap.     

Ultrastructure of the neurohaemal region of the toad median eminence

Summary According to the internal structure and size of the granules, six types of nerve endings can be distinguished in the toad median eminence: 1. Endings containing mostly dense granules of 600 Å in diameter; 2. Endings containing dense granules of about 800 Å in diameter; 3. Endings which contain dense granules 1,000–2,000 Å in diameter, with the peak at 1,200–1,400 Å; 4. Endings containing granules with a characteristic structure, which differentiate them from the other three types; 5. Scarce endings containing granules 2,000 to 3,800 Å in diameter; and 6. Endings containing only vesicles 400–500 Å in diameter. Types 3 and 4 endings are mainly found in the outer pericapillary zone, and are probably responsible for the strong Gomori-positive reaction observed in this zone. The other four types of endings occur mainly in the inner pericapillary zone, and appear to be Gomori-negative.The probable origins of the different types of endings, and their possible relations with the different releasing factors is discussed.The subendothelial basement membrane has numerous long processes which form a complicated network in contact with all the nerve endings, some nerve fibres and glial cells.Two types of glial cells are described. Pinocytotic vesicles are frequently seen at the points where these cells contact the basement membrane. All the ultrastructural features suggest that these cells are carrying out transport functions.Fellow of the Consejo Nacional de Investigaciones Científicas y Técnicas de la República Argentina.The author is very grateful to Professor H. Heller for his continued encouragement and criticism and to Mr. J. Lane and Mr. P. Heap for their valuable help.     

Horizontal reactor for the continuous production of ethanol by yeasts immobilized in pectin

Summary Yeast cells (Saccharomyces cerevisiae) were immobilized in pectin gel, incubated 12 h at 30°C and then used for the continuous production of ethanol employing a wedge-shaped horizontal reactor and sugar cane molasses as the carbon source. Under steady state conditions the mean residence time was 1.6 h and the volumetric productivity 40 g EtOH/hl. The gas evolved was easily released. Successive batch incubation in a synthetic medium substantially restored the fermentative capacity of the beads already used in the continuous assay.Departamento de Biotecnología y Bioingeniería, Centro de Investigación y de Estudios Avanzados del IPN, México D.F.Member of the Scientific Researcher's Career of the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina.     

The effects of adenohypophysectomy on the hypothalamic-hypophysial neurosecretory system and the adrenal gland of the toad Bufo arenarum Hensel

Summary The effects of adenohypophysectomy were studied on the hypothalamichypophysial neurosecretory system and the adrenal glands of Bufo arenarum Hensel. An increase in vascularization of the pars intermedia was found and the neurosecretory material (NSM) in the glandular region of the median eminence disappeared. Its reappearance later was accompanied by hypertrophy of the pars intermedia; differentiated chromophil cells appeared in the pars intermedia around the vessels and, at the same time, the adrenal reverted to normal. These findings are interpreted as hypertrophy and differentiation of the pars intermedia with replacement, at least partly, of the functions of the pars distalis. The probable mechanism of differentiation and the functions of chromophil cells of the pars intermedia are discussed.This paper was presented at the VI Reunión de la Asociación Latinoamericana de Ciencias Fisiológicas (A.L.A.C.F.), Viña del Mar, Chile, 1964. It was carried out under the auspices of the Consejo Nacional de Investigaciones Científicas y Técnicas (Argentina) and the Rockefeller Foundation (School grant RF-58028).Fellow of the Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina.The authors wish to thank Prof. M. H. Burgos for his constant interest, Prof. B. A. Houssay, Prof. H. Heller and Dr. J. H. Tramezzani for their criticisms, Miss B. Rodriguez and Mr. L. Castro for their technical assistance.     

Neurosecretory activity in supraoptic nucleus of normal rats

Summary The secretory rhythm of neurosecretory cells was studied in supraoptic nuclei of normally drinking rats. Both nuclei were divided in five zones, and in each one, the nuclear and nucleolar volumes of 50 neurons were measured. Parallel observations were made on the Nissl material and neurosecretory granules. Comparative effects of different fixatives showed Carnoy and Zenker to be the best fluids for this type of investigation.Two extreme types of neurosecretory neurons with intermediary stages are observed. Type I neuron has a small nucleus and nucleolus, well developed Nissl bodies and a greater content of neurosecretory material. Type II has larger nucleus and nucleolus, scanty or packed Nissl bodies, and little Gomori positive material. The nuclear and nucleolar volumes differ between the different zones of the supraoptic nucleus and the zone of maximal activity varies in each particular case. To explain this asynchronic behavior a neurosecretory wave of activity moving along the supraoptic nucleus is postulated.Supported by Grants of the Consejo Nacional de Investigaciones Científicas y Técnicas, and the Air Force Office of Scientific Research 314-64.Fellows of the Consejo Nacional de Investigaciones Cientificas y Técnicas.     

The secretory cycle of supraoptic neurons in the rat

Summary After perfusion with formaldehyde and glutaraldehyde the supraoptic nucleus and infundibular process of the neurohypophysis of the rat were dissected and prepared for electronmicroscope observation. This study was carried out in specimens under normal water balance, in others fed on dry food and in rats submitted to forced hydration.Two extreme types of neurons with intermediary stages were recognized in the normal supraoptic nucleus. The main difference between them is in the content of ribosomes, development and dilatation of the vacuolar system and in the number of elementary neurosecretory granules. In both types lysosome-like particles are observed. The volume of the elementary granules increases 1.7 times along the hypothalamic-hypophyseal tract while the increase in the dense core of the granule is of the order of 4.3 times.After forty-eight hours on dry food there is a general depletion of secretory granules from the perikaryon and nearby axons, the ribosomes are numerous and the endoplasmic reticulum is dilated in all cells and contains a macromolecular filamentous material. With more prolonged dehydration the neurosecretory granules reappear in relation to the Golgi complex and the vacuolar system becomes progressively flattened. With forced hydration the number of granules in the perikaryon increases considerably.These observations are interpreted as indicative that the early stages of synthesis take place at the level of the ribosomes. The product, in a dilute macromolecular form, is transferred into the cisternae of the endoplasmic reticulum and then condensed into granules within the Golgi complex. The increase in size of the granules along the axon is discussed in relation to the progressive increase in hormone content.Supported by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas and by the Air Force Office of Scientific Research No 963-65.Fellow of the Consejo Nacional de Investigaciones Científicas y Técnicas.