Investigations on the hypothalamo-neurohypophysial neurosecretory system of the grass frog (Rana pipiens) after transection of the proximal neurohypophysis

Summary With the onset of degeneration of the neurosecretory nerve fibers following transection of the proximal neurohypophysis, the pituicytes phagocytize these nerve fibers. Concomitant with a considerable increase in the size of the pituicytes, which reaches a peak between 8 and 10 days after the transection, the following sequence of events can be observed: reduction of the amount of intergranular axoplasm, increase in the size of some granules, partial or total loss of the electron density of the neurosecretory granules, loss of granule membranes, fusion of some granules, polymorphous axonal content in digestion vacuoles, formation of multilamellate bodies, digestion vacuoles with moderately electron dense peripheral material, empty digestion vacuoles. At about 12 days after the transection many vacuoles appear which subsequently disappear as the pituicytes shrink. Free neurosecretory granules resulting from the disappearance of the axolemma remain intact in the intercellular and perivascular connective tissue spaces and are eventually phagocytized by pituicytes and pericytes.Phagocytosis is considered to be a basic function of pituicytes. The problems related to this function as well as the possible implications for the interpretation of Herring bodies are discussed.This investigation was supported by Grant No. NB-06641 of the National Institute of Neurological Diseases and Blindness. The technical assistance of Mrs. Mildred Floyd and Christa Cooper is gratefully acknowledged.Dedicated to Prof. Dr. H. Grau on the occasion of his 70th birthday.     

Ultrastructural changes of the neurohypophysis of the rat after castration

Summary The infundibular processes of the neurohypophysis of male and female rats were studied after different periods of castration. After seven days an increase in neurosecretory granules was observed. Two types of neurosecretory nerve endings were identified: dark ones, with dense neurosecretory elementary granules of 1600 A, and clear ones, with lighter neurosecretory granules of 1800 A. Protoplasmatic pituicytes showed a large increase in lipid granules together with a general hypertrophy. After one week of castration but with hormonal therapy the protoplasmatic pituicytes appeared normal or even showed less lipid granules than in the controls.With one month of castration the changes already mentioned in the nerve endings and pituicytes were more pronounced and after six months even more accentuated. Two types of neurosecretory nerve endings were clearly identified and the protoplasmatic pituicytes were loaded with lipid granules.The probable significance of the two different neurosecretory axons was discussed in relation to recent studies on the isolation of neurosecretory terminals from the neurohypophysis. The changes in the protoplasmatic pituicytes were considered in relation to the possible significance of the lipid granules.Supported by grants from the Consejo Nacional de Investigaciones Cientificas y Técnicas and by the Air Force Office of Scientific Research (AF-AFOSR 963-67).We are deeply indebted to Mrs. Defilippi-Novoa and Mr. Alberto Saenz for their skillful assistence.Associated Investigator, Consejo Nacional de Investigaliones Científicas y Técnicas, Argentina.     

Adaptation of Camelus dromedarius pars nervosa of the hypophysis to winter and summer living conditions

The aim of this work is to study the characteristics of the dromedary nervous lobe and determine how the seasons condition its organization. To this end, electron microscopy was performed and examined quantitatively on animals from winter and summer periods. The results show a higher number of cells in the nervous lobe in summer than in winter. The most abundant glial elements in winter are light pituicytes engulfing neurosecretory nerve fibers making neuroglial contact, and dark pituicytes containing numerous heterogeneous light bodies. In summer, the most distinctive glial cells may be pituicytes in a phagocytic state making contact with characteristic large light bodies that could represent a degenerative process of large neuropeptide storage. Granular pituicytes were also observed in contact with glial and neuronal components. However, lipid droplets, described in pituicytes of other mammals, were not observed in our samples. Quantitative analysis of neurovascular contacts revealed that the number of nerve terminals contacting the basal lamina did not differ between summer and winter, but the mean number of glial processes increased in winter. Our data provides evidence that the storage of neuropeptides is very marked in summer and that, associated with an autophagic and phagocytic phenomenon, this suggests an adaptation to anticipate any situation that would cause dehydration of the dromedary. Thus, in its tough environment, the animal remains permanently prepared to avoid any large water loss.     

Morphology of the neural lobe of the hypophysis in rats treated with furosemide. II. Pituicytes

Following three days of furosemide administration, the characteristics of the pituicytes in the neural lobe of the rat hypophysis were studied. The pituicytes of the animals which were treated with furosemide and had access to water during the treatment showed scarce cytoplasm organelles, conferring them with a clear appearance (light pituicytes), and a frequent finding was neurosecretory axons enclosed in their own cytoplasm. In the animals deprived of water during treatment, the pituicytes were dense (dark pituicytes) elongated, with a well-developed Golgi apparatus, numerous profiles of smooth endoplasmic reticulum and frequent annular gap junctions. It is concluded that the dehydration induced by the diuretic agent is the cause of the prevalence of the dark pituicytes.     

Ultrastructural changes and proliferation of pituicytes in mouse posterior lobe during water deprivation and rehydration

Ultrastructural changes and proliferation of pituicytes during water deprivation and rehydration were studied in the posterior lobe of C57BL/Tw mice. Deprivation for 3 days brought about a significant increase in the number of electron-dense bodies (lysosomes) in pituicyte perikarya and their processes. The frequency of pituicytes enclosing neurosecretory axons in their cytoplasm significantly decreased as compared with that of the controls. 12-hour rehydration following deprivation for 3 days induced extensive development of rough endoplasmic reticulum and Golgi apparatus and an increase in frequency of neurosecretory axons enwrapped by pituicyte cytoplasm. However, at 2 days of rehydration the morphology of pituicytes was no more different from that of the controls. Mitotic figures of pituicytes were not encountered throughout the deprivation period of 6 days, but rehydration for 12 h and 1, 2, or 3 days following deprivation for 3 or 6 days was effective in eliciting an increase in mitotic activity. The present results indicate that pituicytes in the mouse posterior lobe are intimately related with the secretory mechanism of neurosecretory material from the neurosecretory axons and that the proliferation of pituicytes is stimulated in conditions of reaccumulation of neurosecretory material.     

Ultrastructure of the developing gizzard epithelium in the chick embryo

Summary The fine structure of the gizzard epithelium is described at different stages in the development of the chick embryo. The elaborate apical processes, a characteristic feature, take part in secretion at thirteen days, but do not seem to have this function at nine and ten days. The formation of glands begins at thirteen days but the adult fine structure of the gland cells is not attained until hatching. The distinct surface layer present between thirteen and seventeen days may have a protective function. Acknowledgement. The author is grateful for research facilities provided by Professor G. M. Wyburn, Anatomy Department, The University of Glasgow.     

Golgi-like immunostaining of pituicytes and tanycytes positive for glial fibrillary acidic protein in the neurohypophysis of the Mongolian gerbil (Meriones unguiculatus)

Summary Glial cells that contain the glial fibrillary acidic protein (GFAP; the major protein constituent of glial filaments) were stained immunohistochemically in thick frozen sections of the neurohypophysis of the Mongolian gerbil (Meriones unguiculatus). The resulting Golgi-like images provided informations on cytological features and distributional patterns of tanycytes and pituicytes. In the proximal median eminence, numerous bundled processes of tanycytes were revealed. They emerged from the ependymal and subependymal layer and mostly reached the brain surface. Several tanycytic processes extended into the anatomical neural stalk. In the whole neural lobe, a dense network of GFAP-immunoreactive pituicyte processes was visualized. Stained pituicytes were highly asymmetric and exhibited a great morphological variability. Immunopositive fibers which were encountered in the intermediate lobe might be derived from pituicytes. Electron-microscopically further evidence was obtained that GFAP-positive pituicytes correspond to filament-rich fibrous pituicytes at the ultrastructural level.     

Transient expression of a calcium-binding protein (spot 35-calbindin) and its mRNA in the immature pituicytes of embryonic rats

Summary Spot 35 protein is a Ca-binding protein originating from the rat cerebellum; it is now referred to spot 35-calbindin. This protein is expressed in immature pituicytes of the neurohypophyseal anlage in the E11–E18 rat embryo. The gene expression of spot 35-calbindin was detected by in-situ hybridization analysis only at stage E11–E12. Profiles of spot 35-positive nerve fibers of a neurosecretory nature were found in anlage at stage E16. At this stage, some immature pituicytes are partially immunopositive for spot 35-calbindin only in their peripheral cytoplasm; others are immunonegative. At birth and thereafter through adulthood, abundant nerve fibers are the sole structures immunoreactive for spot 35-calbindin; all the pituicytes are immunonegative, resulting in a light-microscopic appearance of numerous immunonegative round profiles, corresponding to pituicytes, and capillaries embedded in the granularly immunostained neurohypophysis. The present findings suggest that, during specific embryonic stages, immature pituicytes exert some as yet unidentified roles related to Ca-mediated functions involving the expression of spot 35-calbindin.     

QUANTITATIVE LYSOSOMAL ENZYME ACTIVITY CHANGES IN THE NEURAL LOBE OF THE RAT FOLLOWING WATER DEPRIVATION AND LACTATION

—The activities of five lysosomal enzymes, acid phosphatase, β-glucosidase, β-glucuronidase, β-galactosidase and N-arylamidase (classified according to Marks (1970)) were measured by means of sensitive microchemical techniques in frozen-dried rat neural lobe tissue after experimental and physiological stimulation of hormone release from the hypothalamo–neurohypophysial system i.e. water deprivation (3 and 6 days), delivery and lactation (10 days). During all conditions of stimulation increases of 29 to 106 per cent were measured for lysosomal enzyme activity, expressed as mmol/ng DNA/h. With histochemical staining methods the acid phosphatase activity appears to be mainly localized in the pituicytes, but it was impossible to visualize the microchemically measured acid phosphatase activity increase within the two main compartments of the neurohypophysis, i.e. axonal endings and the neurohypophysial glial cells, the pituicytes.     

Endogenous β-d-galactoside binding lectin during the expansion of the yolk sac in the developing chick embryo

Summary A lectin with an affinity for -d-galactoside-containing saccharides is present in the developing yolk sac from the chick embryo at stages from 2 to 7 days of incubation. This activity is present in the area vitellina (less differentiated) and the area vasculosa (more differentiated). In both areas, lectin activity increases significantly during the spreading of the yolk sac up to 5 days of incubation. At all of the stages studied lectin activity was significantly higher in the area vasculosa, as compared to the area vitellina.Lectins were purified by affinity chromatography and examined by SDS-PAGE. Under reducing conditions two components are evident. A more prominent band of subunit molecular weight of 14,200±100 for the area vitellina and 13,700±300 for the area vasculosa and a second band with molecular weight of about 68,000±700 and 68,000±1,200 for the area vitellina and area vasculosa respectively, were observed. The -d-galactoside-binding lectin appears to be similar if not identical to that of the early chick blastoderm.     

Urea: a nitrogen source for the aquatic resurrection plant Chamaegigas intrepidus Dinter

Chamaegigas intrepidus Dinter is a poikilohydric aquatic plant that lives in rock pools on granite outcrops in central Namibia. The pools are filled with water only intermittently during the wet season, and the plants may pass through up to 20 rehydration/dehydration cycles during the summer rains. The potential nitrogen sources for the rehydrated plants are ammonium, which is only present at 10–20 μm, amino acids, particularly glycine, and urea, which is generally present at 20–30 μm. We show that urea can be utilised by plants in the field through the presence of urease in the sediments of the rock pools. Urease activity is higher in non-submerged than in submerged sediments, and it can survive 6 months of complete dryness at temperatures up to 60°C. Experiments with [¹⁴C]urea under laboratory conditions show that the roots of C. intrepidus are unable to take up urea; while ¹⁵N-nuclear magnetic resonance experiments show that [¹⁵N]urea is only metabolised to labelled glutamine and glutamate after ammonium has been released by the action of urease. Thus urease plays a vital role in allowing urea to be utilised as a major N source in this nutrient-limited aquatic ecosystem. Received: 23 April 1999 / Accepted: 8 November 1999     

Elektronenmikroskopische Untersuchungen über die Reaktion der Pituizyten nach Hypophysenstieldurchtrennung bei Rana esculenta

Zusammenfassung Etwa 2 Tage nach Durchtrennung des Hypophysenstiels beginnen die Pituizyten der Froschhypophyse (Rana) die degenerierenden sekrethaltigen Axone zu phagozy tieren. Nach 3–4 Tagen werden die Neurosekretgranula in den Einschlußkörpern immer mehr zusammengedrängt und verlieren schließlich ihren elektronendichten Inhalt. Die Hüllen der Elementargranula verwandeln sich mit eingelagerten Lysosomen zu membranösen, sehr stark osmiophilen Körpern, die sich schließlich langsam auflösen.Die mitotische Aktivität der Pituizyten ist während des Abbaues gesteigert. Zwischen Gefäßen und Pituizyten bestehen enge räumliche Beziehungen. Die Fähigkeit zur Phagozytose wird als Grundfunktion der Pituizyten angesehen.

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Electron microscopical investigation on the reaction of the pituicytes after stalk section in Rana esculenta

Summary About 2 days after transsection of the pituitary stalk the pituicytes of the frog neurohypophysis (Rana) start to phagocytate the degenerating neurosecretory axons. After 3–4 days the neurosecretory granules inside the inclusion bodies are compressed more and more and finally their electron-dense contents disappear. The membranes of the elementary granules together with lysosomes alter into membranous highly osmiophilic bodies slowly dissolving at last.During the desintegration of nerve endings the mitotic activity of the pituicytes increases. Blood vessels and pituicytes are closely connected.It is supposed that the ability of phagocytosis represents a basic function of the pituicytes.

     

The fine structure of the retinal plexus in Octopus vulgaris

Summary The plexiform layer of the octopus retina was investigated by means of electron microscopy. The axonic processes of visual cells contain closely packed microtubules, 300 Å in diameter, running parallel to one another along the long axis of the processes. Visual cells also send out a large number of thin axon collaterals. Each of them forms presynaptic knobs with numerous clear vesicles along their course. These are assumed to be concerned with the reciprocal communication between visual cells. Nerve endings with dense-cored vesicles form synaptic contacts with visual cells. The visual cells show some spherical protrusions into the perivascular spaces.Octopuses, captured off the coast of Onagawa (Miyagi-ken, Japan) in autumn of 1963 and 1964, were offered for this research through the kindness of Dr. Kyoji Tasaki, Assistant Professor of Physiology in Tohoku University School of Medicine.I wish to thank Professor Toshiyuki Yamamoto for his encouragement and suggestions throughout all stages of this work, and also Mr. Masae Kato for his technical assistance in drawing.     

Isolation and characterization of a d-glucose/xylose isomerase from a new thermophilic strain Streptomyces sp. (PLC)

A thermostable d-xylase isomerase from a newly isolated thermophilic Streptomyces sp. (PLC) strain is described. The enzyme was purified to homogeneity. It is a homotetramer with a native molecular mass of 183 kDa and a subunit molecular mass of 46 kDa. The enzyme has a K _m of 35 mM for d-xylose and also accepts d-glucose as substrate, however, with a tenfold higher K _m (0.4 M) and half the maximum velocity. Both the activity and stability of this d-xylose isomerase depend strongly on divalent metal ions. Two metal ions bind per subunit to non-identical sites. Mg²⁺, Mn²⁺ and Co²⁺ are of comparable efficiency for the d-xylose isomerase reaction. Con²⁺ is the most efficient cofactor for d-glucose isomerization. The enzyme remains fully active up to 95°C. The activity decreases at 53°C in the presence of Co²⁺ and Mg²⁺ with a half-life of 7 and 9 days respectively. In the presence of Mn²⁺ the enzyme activity remains constant for at least 10 days and at 70°C 50% of the activity is lost after 5 days.     

Glial fibrillary acidic protein and differentiation of neonatal rat pituicytes in vitro

Summary The appearance and intracellular localisation of glial fibrillary acidic protein (GFAP) in pituicytes in neural lobe cultures of newborn rats aged 7 to 30 days were investigated by use of the indirect immunofluorescence method. GFAP-immunoreactive cells were observed mostly in the outgrowth zone. GFAP was localised in the perikaryal cytoplasm as well as in pituicyte processes. GFAP-positive pituicytes showed considerable morphological polymorphism. The presence of GFAP — astrocytic marker — in pituicytes in vitro and the evident morphological similarity to cultured astrocytes suggest the astroglial character of these cells.     

Beiträge zur Taxonomie der Rhizobiaceae

Zusammenfassung Durch Anwendung der DNA-DNA-Hybridisierungsmethode von McCarthy u. Bolton (1963) werden verwandtschaftliche Beziehungen zwischen der Gattung Agrobacterium und Rhizobium nachgewiesen. Außerdem ergeben sich Hinweise, die taxonomische Affinitäten einiger Blattsymbionten in Rubiaceae zu den Rhizobiaceae vermuten lassen.

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Contribution to the taxonomy of the rhizobiaceae

Summary By application of the DNA-DNA-hybridizationmethod (McCarthy and Bolton, 1963) relatedness between the genera Agrobacterium and Rhizobium is demonstrated. Some indications show taxonomic affinities of symbionts in leaves of Rubiaceae to the Rhizobiaceae.

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Herrn Prof. Dr. F. Schwanitz zum 60. Geburtstag gewidmet.     

Purification and properties of D-gluconate dehydratase fromAchromobacter

d-Gluconate dehydratase fromAchromobacter, grown ond-gluconate, was purified 100-fold by a procedure involving ammonium sulfate fractionation and preparative acrylamide gel electrophoresis. The purified enzyme appeared to be homogeneous by disc gel electrophoresis. It is an inducible enzyme with an optimal activity in the pH region 8.4–8.8, a Km value of 2.08 × 10^–2 m ford-gluconate and a molecular weight of 270,000 ± 25,000. Only C₅ and C₆ aldonic acids possessing al-threo configuration at C₂ and C₃ are dehydrated. The dehydration products ofd-gluconate,d-xylonate,d-galactonate,d-fuconate andl-arabonate were identified as 2-keto-3-deoxy compounds by specific colour reactions and thin layer chromatography. Onemm Mg^{+ +} is a powerful activator, 0.1 mm Mn^{+ +} activates poorly and EDTA inhibits. Glutatione, dithiothreitol and mercaptoethanol had no effect, althoughp-chloromercuribenzoate (0.01 mm) decreased enzyme activity.We wish to thank Mr D. Dewettinck for skilful technical assistance. The senior author (J.D.L.) is indebted to the Fonds voor Kollektief en Fundamenteel Onderzoek (Belgium) for research and personnel grants. J.K.-M. is indebted to the Belgian government for a travel and study grant.     

Effect of acetyl-l-carnitine on lipid peroxidation and xanthine oxidase activity in rat skeletal muscle

It has been reported that acetyl-l-carnitine (AcCn) can reduce the degenerative processes in the central nervous system of rats, modify the fluidity of membranes and decrease the accumulation of lipofuscins in neurones. In light of these considerations we have assayed the in vitro effect of acetyl-l-carnitine on spontaneous and induced lipoperoxidation in rat skeletal muscle; in addition, the effect of AcCn on XD/XO ratio was evaluated. The presence of AcCn (10–40 mM) in incubation medium significantly reduced MDA and conjugated diene formation in rat skeletal muscle; moreover, a significant decrease in induced MDA levels was observed when microsomal preparation where incubated in the presence of 10–40 mM AcCn. Since a significant reduction of XO activity was detected in the presence of 10–80 mM AcCn, the reduced lipid peroxidation by AcCn seems to be due to an inhibition of XO activity.     

Observations on neurons and neuroglia from the area of the reticular formation in tissue culture

Summary The area of the reticular formation of the brain stem of several newborn and young manmals was cultivated in vitro. Neurons were observed for a period up to 54 days. At the end of this period in many preparations they still exhibited signs of chromatolysis as judged by the eccentric position of the nuclei and the peripheral location of the Nissl material. It has been impossible thus far to identify particular neurons as belonging to a particular nucleus of the reticular formation, probably due to slight morphological and structural changes of these cells in vitro. Although the oldest neurons observed were kept only 54 days in vitro it seems likely that they could have been maintained for a longer time since their appearance indicated more regenerative rather than degenerative features.Observations on the morphology and kinetics of glial and mesenchymal cells corroborated the findings of Pomerat and Costero (1956) and of Hild (1954, 1957a).This investigation was supported by a research grant (PHS B-364 [C4]) from the National Institute of Neurological Diseases and Blindness, of the National Institutes of Health, Public Health Service, administered by C. M. Pomerat.Fulbright and Smith-Mundt fellow.     

Metabolic pathways and biotechnological production of l-cysteine

l-Cysteine is an important amino acid both biologically and commercially. Although most amino acids are commercially produced by fermentation, cysteine is mainly produced by protein hydrolysis. However, synthetic or biotechnological products have been preferred in the market. Biotechnological processes for cysteine production, both enzymatic and fermentative processes, are discussed. Enzymatic process, the asymmetric hydrolysis of dl-2-amino-Δ²-thiazoline-4-carboxylic acid to l-cysteine, has been developed and industrialized. The l-cysteine biosynthetic pathways of Escherichia coli and Corynebacterium glutamicum, which are used in many amino acid production processes, are also described. These two bacteria have basically same l-cysteine biosynthetic pathways. l-Cysteine-degrading enzymes and l-cysteine-exporting proteins both in E. coli and C. glutamicum are also described. In conclusion, for the effective fermentative production of l-cysteine directly from glucose, the combination of enhancing biosynthetic activity, weakening the degradation pathway, and exploiting the export system seems to be effective.