Electron microscopy of taste buds of the rat

Summary The taste buds of the circumvallate papillae have been examined by electron microscopy in OsO₄-fixed, PTA stained material or after KMnO₄ fixation. The microvilli of the receptor cells have terminal dilatations which presumably give an increased surface area for transduction. The extracellular spaces at the necks of the receptor cells near the bases of the microvilli are interrupted by closed contacts.The synapses have a well defined synaptic cleft suggesting a chemical rather than an electrical mode of transmission. Synaptic membrane specialisations differ from the membrane thickenings of other types of synapse. Presynaptic dense projections are present but there is no well define postsynaptic thickening. Vesicles occur in both pre- and postsynaptic components, but it is debatable whether or not they should be termed synaptic vesicles. Acknowledgements. We are indebted to Professor J. Z. Young, F. R. S., for his stimulating support, and to Mr. S. Waterman for skilled photography.     

Displacement of excitatory amino acid receptor ligands by acidic oligopeptides

A number ofD-glutamyl andL-aspartyl dipeptides, glutathione, -D-glutamylglycine and -D-glutamyltaurine, were tested for their efficacy to displace ligands specific for different subtypes of excitatory amino acid receptors from rat brain synaptic membranes. In general, theL enanthiomorphs of -glutamyl peptides were more potent displacers than -D-glutamylglycine and-taurine but the latter were more specific for the quisqualate type of receptors. -L-glutamyl-L-glutamate was the most effective dipeptide in displacing the binding of glutamate, 2-amino-3-hydroxy-5-methylisoxazole-4-proprionate (AMPA) and 2-amino-5-phosphonoheptanoate (APH), whereas -L-glutamyl-L-aspartate was the most effective in the binding of kainate. Both oxidized and reduced glutathione were inhibitory, being most potent in the binding of AMPA. -L-Glutamylaminomethylsulphonate was most effective in the binding of APH. The most potent -L-glutamyl peptides (glutathione, -L-glutamyl-L-glutamate,-L-aspartate, and-glycine) may act as endogenous modulators of excitatory aminoacidergic neurotransmission.     

Cholinergische Synapsen im Oberschlundganglion der Waldameise (Formica lugubris Zett.)

Summary The corpora pedunculata of the wood ant (Formica lugubris Zett.) consist of two sharply defined layers: The perikaryon layer and the subjacent neuropil. Synaptic endings are found exclusively in the neuropil. The synapses consist of a central, presynaptic end knob of 1.2–2.5 diameter and a relatively large number of surrounding postsynaptic processes of 0.3–1.1 diameter. These junctions are analogous to axodendritic synapses of the vertebrates. The presynaptic process contains mitochondria and a multitude of light vesicles (300–600 Å diameter). Larger vesicles 700–1000 Å with a dark center are seen more rarely. The synaptic cleft has a diameter of approximately 130 Å and varies somewhat with different fixation methods. With glutaraldehyde-osmium fixation, this relatively wide gap is maintained only in circumscribed areas of the junction, while in adjacent areas it tends to contract and an external compound membrane is formed. The postsynaptic region is characterized by the presence of a subsynaptic network which is revealed only by suitable fixation methods. This and the persistent synaptic cleft are the main structural differentiations found in junctional areas thus far.Cholinesterase is located with the aid of thiolacetic acid (Barnett) and Eserin control studies. The enzyme is found in the cytoplasm immediately adjacent to the pre- and postsynaptic membranes. In two thirds of our observations the reaction is far more concentrated postsynaptically than presynaptically. In one third, the distribution is reversed. Only an insignificant amount of cholinesterase is present within the synaptic cleft. There is no evidence that cholinesterase is evenly distributed along the entire junctional region. In contrast, only small circumscribed areas show a positive reaction and these coincide with the extent of the synaptic cleft and the subsynaptic network. Such areas seem to correspond to the active junctional areas.

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Unterstützt durch einen Kredit (Nr.2575) des Schweizer Nationalfonds für Wissenschaftliche Forschung.

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Herrn Prof. Dr. W. Bargmann zum 60. Geburtstag gewidmet.     

A quantitative study of synaptic interconnections in the dorsal lateral geniculate nucleus of the cat

Summary 2,700 synaptic contacts have been classified according to criteria described in an accompanying paper and the results summarized in tabular form. Only about 20% of the synaptic contacts in laminae A and A1 are formed by axons identifiable as retinogeniculate fibers. About 1/4 of these retinogeniculate synapses are axo-axonal. Approximately 45% of the contacts in these laminae are formed by axons tentatively identifiable as corticogeniculate fibers; about 35% by presumed intrageniculate fibers. Close to one half of the synapses occur in encapsulated synaptic zones, where grapelike dendritic appendages are related mainly to intrageniculate and retinogeniculate axons, and about half lie in interstitial zones, where corticogeniculate and some intrageniculate axons contact distal dendritic segments.Regions of the nucleus receiving from peripheral parts of the retina have relatively more corticogeniculate synapses, and have fewer intrageniculate synapses in the encapsulated zones than do regions receiving from the central parts of the retina.Most of the tissue in lamina B resembles the interstitial zones of laminae A and A1 and over 2/3 of the contacts in lamina B may prove to be corticogeniculate. The retinogeniculate fibers in this lamina are associated with relatively few other axons in simple, small encapsulated zones.Supported by Grant NB 06662 from the USPHS. The skillful technical assistance given by Mrs. E. Langer during the course of this work is gratefully acknowledged.     

Synaptic organization of the cabbage looper moth ocellus

Summary Chemical and electronic synapses are present in the ocellar synaptic region of the moth, Trichoplusia ni. The chemical synapses all appear to be of the conventional type. Four different chemical synaptic contacts were observed: Receptor cell axons presynaptic to receptor cell axons, receptor cell axons presynaptic to 1st order interneurons, 1st order interneurons presynaptic to receptor cell axons, and 1st order interneurons presynaptic to 1st order interneurons. Two different types of contact made by electronic synapes were observed: Contacts between receptor cell axons and 1st order interneurons, and contacts between 1st order interneurons. The significance of this synaptic arrangement for the generation of on and off responses in the 1st order interneurons is discussed.Supported by NSF Grant BMS 75-07645 and by the VPI & SU Research Division     

An ultrastructural study of the carotid body of horse and dog

Summary Carotid body tissue from horse and dog has been investigated ultrastructurally. Several cell types are recognized: glomus cells which are regarded as chemoreceptors, sustentacular cells which enclose the glomus cells, and nerve fibers.The glomus cells contain electron dense granules which are interpreted as packages of biogenic monoamines. There are both dark and light glomus cells, the former containing more granules and ribosomes. Invaginations of the plasma membranes as well as free coated vesicles are often seen in the cytoplasm of glomus cells. Nerves within the glomus lobules are generally wrapped by sustentacular cells, but nerve endings are also seen in close contact with the glomus cells. Some endings contain synaptic vesicles as well as a great concentration of mitochondria. The corresponding fibers are thought to be efferent. Another type of contact is interpreted as en passant synapses of afferent fibers.The author wishes to express his gratitude to Professor L. Nicander who initiated this project and took most of the micrographs and to Professor Nils Obel and associate Professor Gustav Björk at the Royal Veterinary College for their valuable help with the surgical procedure and to Dr. Martin Ritzén of the Royal Medical College for making the tests for biogenic monoamines.     

Properties of Aspergillus japonicus β-fructofuranosidase immobilized on porous silica

-Fructofuranosidase from Aspergillus japonicus MU-2, which produces fructo-oligosaccharides (1-kestose: O--D-fructofuranosyl-(2 1)--D-fructofuranosyl -D-glucopyranoside); and nystose: O--D-fructofuranosyl-(2 1)--D-fructofuranosyl-(2 1)--D-fructofuranosyl -D-glucopyranoside) from sucrose, was immobilized, covalently with glutaraldehyde onto alkylamine porous silica, at high efficiency (64%). Optimum pore diameter of porous silica for immobilization of the enzyme was 91.7 nm. After immobilization, the enzyme's stabilities to temperature, metal ions and proteolysis were improved, while its optimum pH and temperature were unchanged. The highest efficiency of continuous production of fructo-oligosaccharides (more than 60%), using a column packed with the immobilized enzyme, was obtained at 40% to 50% (w/v) sucrose. The half-life of the column during long-term continuous operation at 55°C was 29 days.     

Special somatic spine synapses in the ciliary ganglion of the chick

Summary Somatic spine synapses modified with postsynaptic electron opaque materials were found in the axo-somatic ciliary ganglion synapse of the chick.A part of the postsynaptic cell body protrudes into the presynaptic calyciform ending as a somatic spine with about 1 in length and 0.15 in diameter, and forms the so-called synaptic complex with presynaptic process. Moreover, conspicuous electron opaque materials can be seen in the central axis of the spine, except for its end portion. Sometimes, these opaque materials are seen as arrayed dots.The morphological characteristics of the somatic spine synapses in this study are quite similar to that found in the habenula and interpeduncular nuclei of the cat (Milhaud and Pappas, 1966). the biological significance of which is obscure at present.This work was supported in part by grant from the Education Ministry of Japan.     

Purification and characterization of α(2-6)-sialyltransferase from human liver

A Gal1-4GlcNAc (2-6)-sialyltransferase from human liver was purified 34 340-fold with 18% yield by dye chromatography on Cibacron Blue F3GA and cation exchange FPLC. The enzyme preparation was free of other sialyltransferases. It did not contain CMP-NeuAc hydrolase, protease, or sialidase activity, and was stable at –20°C for at least eight months. The donor substrate specificity was examined with CMP-NeuAc analogues modified at C-5 or C-9 of theN-acetylneuraminic acid moiety. Affinity of the human enzyme for parent CMP-NeuAc and each CMP-NeuAc analogue was substantially higher than the corresponding Gal1-4GlcNAc (2-6)-sialyltransferase from rat liver.Abbreviations FPLC fast protein liquid chromatography - NeuAc 5-N-acetyl-d-neuraminic acid - 9-amino-NeuAc 5-acetamido-9-amino-3,5,9-trideoxy-d-glycero-2-nonulosonic acid - 9-acetamido-NeuAc 5,9-diacetamido-3,5,9-trideoxy-d-glycero--d-2-nonulosonic acid - 9-benzamido-NeuAc 5-acetamido-9-benzamido-3,5,9-trideoxy-d-glycero--d-galacto-2-nonulosonic acid - 9-fluoresceinyl-NeuAc 9-fluoresceinylthioureido-NeuAc - 5-formyl-Neu 5-formyl--d-neuraminic acid - 5-aminoacetyl-Neu 5-aminoacetyl--d-neuraminic acid - CMP-NeuAc cytidine-5-monophospho-N-acetylneuraminic acid - G_M1 Gal1-3GalNAc1-4(NeuAc2-3)Gal1-4Glc-ceramide - ST sialyltransferase - DTE 1,4-dithioerythritol Enzyme: Gal1-4GlcNAc (2-6)-sialyltransferase, EC 2.4.99.1.     

Location by fluorescence microscopy of glycosidases and a xylanase in the anaerobic gut fungi Caecomyces communis,Neocallimastix frontalis,and Piromyces rhizinflata

-d-Glucosidase, -d-fucosidase -d-xylosidase, and -cellobiopyranosidase activities in Caecomyces communis, Neocallimastix frontalis, and Piromyces rhizinflata, located with fluorescent conjugates, occur throughout the whole thallus as from zoospore germination and disappear before sporulation. -d-Galactosidase and -l-arabinopyranosidase activities are low or nonexistent. A xylanase, detected by indirect immunofluorescence, was observed at the surface of the vegetative cells, vesicles, or rhizoids. Cross-reactions prove the existence of analogies in structure among the enzymes of these anaerobic gut fungi.     

Gibberellins play a role in the interaction between imidazole fungicides and cytokinins in Araceae

Imidazole fungicides such as imazalil, prochloraz, and triflurnizole and the triazole growth retardant paclobutrazol promote the shoot-inducing effect of exogenous cytokinins in Araceae, such as Spathiphyllum floribundum Schott and Anthurium andreanum Schott. The mechanism of their action could partially be based on the inhibition of gibberellic acid (GA) biosynthesis, because administration of GA₃ inhibits the phenomenon completely in S. floribundum. Not only is the suppression of GA biosynthesis involved, but also the metabolism of endogenous cytokinins is significantly altered. Although the balance between isopentenyladenine, zeatin, dihydrozeatin, and their derivatives was shifted to distinguished directions by administration of BA and/or imazalil and/or GA₃, no correlation between these changes in metabolic pathways and the number of shoots could be found. The metabolism of BA was not significantly altered by adding imazalil to the micropropagation medium of S. floribundum.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - [9R-5P]DHZ 9--d-ribofuranosyl-dihydrozeatin-monophosphate - [9R-5P]iP 6-isopentenyl-9--d-ribofuranosyladenine-monophosphate - [9R-5P]Z 9--d-ribofuranosyl-zeatin-monophosphate - [9G]BA 6-benzyl-9--d-glucopyranosyladenine - [9G]DHZ 9--d-glucopyranosyl-dihydrozeatin - [9G]iP 6-isopentenyl-9--d-glucopyranosyladenine - [9G]Z 9--d-glucopyranosyl-zeatin - [9R]BA 6-benzyl-9--d-ribofuranosyladenine - [9R]DHZ 9--d-ribofuranosyl-dihydrozeatin - [9R]iP 6-isopentenyl-9--d-ribofuranosyladenine - [9R]Z 9--d-ribofuranosyl-zeatin - BA 6-benzyladenine - DHZ dihydrozeatin - ES⁺ LC-MS/MS HPLC coupled Electrospray Tandem Mass Spectrometry - f.m. fresh mass - mT 6-(3-hydroxybenzyl)adenine - IMA imazalil - iP isopentenyladenine - NAA 1-naphthalene acetic acid - NFT Nutrient Film Technique - (OG)[9R]DHZ O--glucopyranosyl-9--d-ribofuranosyl-dihydrozeatin - (OG)[9R]Z O--d-glucopyranosyl-9--d-ribofuranosyl-zeatin - (OG)DHZ O--d-glucopyranosyl-dihydrozeatin - (OG)Z O--d-glucopyranosyl-zeatin - PAR Photosynthetic Active Radiation - PBZ paclobutrazol - PRO prochloraz - TDZ thidiazuron - TRI triflurnizole - Z zeatin     

Synaptic junction development in the spinal cord of an amphibian embryo: An electron microscope study

Summary An electron microscopical study has been made of the cervical spinal cord of Xenopus laevis embryos, from the time that the neural tube closes until the larvae were hatched and could swim. Sections of the whole cord were searched for intercellular junctions during this period. Two nonsynaptic types were found, the first were widely distributed puncta adherentia, the second were rare and similar to gap junctions. Membrane specializations with synaptic vesicles were first found when the neural folds had fused; membrane-vesicle clusters which looked like the presynaptic half of a synaptic junction were present, together with synaptic junctions lacking any postsynaptic membrane thickening or cytoplasm density. About four hours later, mature synaptic junctions with full thickening of the postsynaptic membrane, dense cytoplasm and striated or dense material in the synaptic cleft were present. Presynaptic mitochondria, dense-cored and flattened vesicles, fibre to fibre and fibre to cell body synapses were present from the first, as were synapses onto very fine dendrites which might be filopodia from dendritic growth cones. Synaptogenesis may start with the accumulation of vesicles in dense cytoplasm near a thickened cell membrane; the postsynaptic element becomes associated with this membrane-vesicle cluster and matures by increasing cleft and cytoplasmic density, and by membrane thickening.     

Substrate specificity and inhibition of UDP-GlcNAc:GlcNAcβ1-2Manα1-6R β1,6-N-acetylglucosaminyltransferase V using synthetic substrate analogues

UDP-GlcNAc:GlcNAc 1-2Man1-6R (GlcNAc to Man) 1,6-N-acetylglucosaminyltransferase V (GlcNAc-T V) adds a GlcNAc1-6 branch to bi- and triantennaryN-glycans. An increase in this activity has been associated with cellular transformation, metastasis and differentiation. We have used synthetic substrate analogues to study the substrate specificity and inhibition of the partially purified enzyme from hamster kidney and of extracts from hen oviduct membranes and acute myeloid leukaemia leukocytes. All compounds with the minimum structure GlcNAc1-2Man1-6Glc/Man-R were good substrates for GlcNAc-T V. The presence of structural elements other than the minimum trisaccharide structure affected GlcNAc-T V activity without being an absolute requirement for activity. Substrates with a biantennary structure were preferred over linear fragments of biantennary structures. Kinetic analysis showed that the 3-hydroxyl of the Man1-3 residue and the 4-hydroxyl of the Man- residue of the Man1-6(Man1-3)Man-RN-glycan core are not essential for catalysis but influence substrate binding. GlcNAc1-2(4,6-di-O-methyl-)Man1-6Glc-pnp was found to be an inhibitor of GlcNAc-T V from hamster kidney, hen oviduct microsomes and acute and chronic myeloid leukaemia leukocytes.Abbreviations all allyl - AML acute myeloid leukaemia - BSA bovine serum albumin - CML chronic myelogenous leukaemia - Gal G,d-galactose - Glc d-glucose - GlcNAc Gn,N-acetyl-d-glucosamine - HPLC high performance liquid chromatography - Man M,d-mannose - mco 8-methoxycarbonyl-octyl, (CH₂)₈COOCH₃ - Me methyl - MES 2-(N-morpholino)ethanesulfonate - oct octyl - pnp p-nitrophenyl - T transferase     

The ovarial enzymes hydrolysing l-leucyl- and dl-alanyl-β-naphthylamide

Summary Fractionation of the rat ovarial tissue homogenate was performed using gel filtration on Sephadex G 200 and by starch gel electrophoresis. The activities hydrolysing l-leucyl--naphthylamide (Leu--NA) and dl-alanyl--naphthylamide (Ala--NA) were determined and partially characterized. Leu--NA was hydrolysed by four separate enzyme activities separated by both methods. Two of them were thiol-activated, one metal-activated and inhibited by EDTA. One was affected by neither metal chelators nor by sulfhydryl reagents. Ala--NA was hydrolysed by the three first-mentioned activities, but not by the last one. In addition, Ala--NA was hydrolysed by two other activities which were totally inhibited by metal chelators. These were clearly separated only using starch gel electrophoresis. The possibilities for the histochemical demonstration of these activities are discussed.     

Synaptische Strukturen und Elementargranula in der Neurohypophyse des Meerschweinchens

Zusammenfassung Die Ultrastruktur des Nucleus infundibularis tuberis, der Zona interna, der Zona externa und des Hypophysenhinterlappens von Meerschweinchen wird beschrieben. Vergleichende Beobachtungen an den Nervenfasern der genannten Gebiete zeitigen folgende Ergebnisse:Die Axone der Zona interna und des Hypophysenhinterlappens enthalten gleichgeartete, große neurosekretorische Elementargranula (1,400 Å). Die Axonquerschnitte im Bereich des Nucleus infundibularis tuberis und der Zona externa weisen Granula gleichen Kalibers (800 Å) auf und lassen damit auf einen funktionell-morphologischen Zusammenhang des Nucleus infundiburis und der Zona externa schließen. Ob die kleinen Elementargranula das morphologische Substrat der sogenannten releasing factors darstellen oder ob es sich um adrenerge Axone und Endigungen mit typischen Katecholamingranula handelt, ist nicht zu klären.Es wird unterschieden zwischen echten Synapsen und synapsenähnlichen Kontakten. Nur erstere zeigen alle klassischen Synapsenmerkmale. Ihnen sind im Bereich der Neurohypophyse axosomatische und interaxonale Synapsen zuzuordnen. Zona externa und Hypophysenhinterlappen stellen neurohämale Kontaktzonen dar und zeichnen sich durch gleichartig konstruierte Synapsen aus. Neben interaxonalen Synapsen treten vor allem neurokapilläre Kontakte in den Vordergrund. In der Zona externa sind die Tanyzyten synaptisch mit Axonen verknüpft, so wie im Hypophysenhinterlappen die Pituizyten synaptische Verbindungen mit Nervenfasern eingehen. Die synapsenähnlichen Bildungen im Verlauf und an den Enden der Axone können somit neurokapillärer, neurotanyzytärer und neuropituizytärer Natur sein. Sie enthalten neben den synaptischen Vesikeln auch meist kleine oder große Elementargranula. Die ähnliche Morphologie der synaptischen Bildungen in der Zona externa und im Hinterlappen läßt einen gleichgearteten Inkretionsmechanismus beider neurohypophysärer Abschnitte vermuten. Tanyzyten und Pituizyten könnten dabei eine Überträgerfunktion ausüben. Eine rezeptorische Funktion der Tanyzyten wird gleichfalls diskutiert.Die Drüsenzellen der Pars intermedia der Hypophyse sind durch direkte neuroglanduläre Kontakte mit den Neuronen des Hypothalamus verknüpft. Die Axone stülpen sich hierbei tief in das Zytoplasma der Drüsenzellen ein. Es entstehen dadurch beim Meerschweinchen drei nervöse Endigungsformen, die Synapsen ausbilden: 1. Endigungen, die nur synaptische Vesikel enthalten; 2. Endigungen mit synaptischen Vesikeln und kleinen Granula; 3. Endigungen mit synaptischen Vesikeln und großen Granula.Zwischen den Drüsenzellen der Pars infundibularis der Adenohypophyse sind ebenfalls Axonquerschnitte zu beobachten. Es handelt sich dabei entweder um vegetative Faserbündel oder Nervenfasern mit kleinen Granula wie in der Zona externa (800 Å).

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Summary The ultrastructure of the nucleus infundibularis tuberis, the inner and outer layer of the median eminence and the posterior lobe of the guinea pig are described. Comparative studies on the nerve fibres of these regions lead to the following conclusions:The axons of the inner layer of the median eminence and of the posterior lobe contain similar types of large, neurosecretory elementary granules (Diameter 1400 Å).The cross-sections of the axons in the area of the nucleus infundibularis tuberis and of the outer layer of the median eminence exhibit the same sized granules (Diameter 800 Å), which points to a functional-morphological relationship between the two. Whether the small elementary granules represent the morphological substrate of the so-called releasing factors, or whether it is a question of adrenergic axons and terminations with typical catecholamine granules, is undecided.A distinction is made between true synapses and synapse-like contacts. Only the former display all the classical characteristics of synapses. To this sort must be classed the axosomatic and interaxonal synapses in the neurohypophysis. The external layer of the median eminence and the posterior lobe are distinguished by neurohaemal contact areas and show similarly constructed synapses. Of particular prominence, besides interaxonal synapses, are neurocapillary contiguities. Just as in the posterior lobe the pituicytes show synaptic connections with nerve fibres, so are the tanycytes in the outer layer of the median eminence in synaptical contact with axons. Thus the synapse-like formations along and at the endings of the axons can be of a neurocapillary, neurotanycytial and neuropituicytial nature. They contain, apart from the synaptic vesicles, mainly small but also large elementary granules. The similar morphology of the synaptic formations in the outer layer of the median eminence and the posterior lobe suggests the existence of a similar mechanism of internal secretion in both these neurohypophysial areas. Tanycytes and pituicytes could exercise a carrier function. A possible receptor-function of the tanycytes is considered.The glandular cells of the pars intermedia of the hypophysis are linked by means of direct neuroglandular contacts with the neurons of the hypothalamus, whereby the axons make deep incursions into the cytoplasm of the glandular cells. In the guinea pig three types of termination can be observed that form synapses: 1. terminations that contain only synaptic vesicles; 2. terminations with small granules; 3. terminations with large granules.Cross-sections of axons can also be observed between the glandular cells of the pars infundibularis of the adenohypophysis. Here they can be identified either as vegetative fibre-bundles or as nerve fibres with small granules, such as occur in the outer layer of the median eminence (Diameter 800 Å).

     

Methylobacterium rhodesianum MB 126 possesses two acetoacetyl-CoA reductases

Two constitutive acetoacetyl-CoA (AcAc-CoA) reductases were purified from Methylobacterium rhodesianum MB 126, an NADPH-linked d(-)--hydroxybutyryl-CoA forming reductase (enzyme A) and an NADH-and NADPH-linked l(+)--hydroxybutyryl-CoA forming reductase (enzyme B). Enzyme A and B give apparent K _m values of 15 M and 30 M for AcAc-CoA, 18 M for NADPH and 30 M for NADH, respectively. They are inhibited by AcAc-CoA at concentrations higher than 25 M and 50 M, respectively. The contribution of the two reductases to poly--hydroxybutyrate synthesis is discussed.     

Azoindoxylverfahren zum Hydrolasennachweis

Zusammenfassung Untersucht wird die Eignung verschiedener Azoindoxyl-methoden zum lichtmikroskopisch-histochemischen Nachweis der -N-Acetylglucosaminidase. Die Inkubationsmedien enthalten 0,5 mg N-Acetyl-(5-bromindol-3-yl)--d-glucosaminid (5-Br-3-Indolyl--d-N-acetylglucosaminid; 1 mg gelöst in 0,05 ml Dimethylformamid) in 1 ml 0,1 M Citronensäure-Phosphat-Puffer, pH 4,5 oder 5. Als Simultankuppler werden 0,02 ml Hexazonium-p-rosanilin oder-neufuchsin oder tetrazotiertes BAXD/ml oder 0,5 mg Fast Blue B oder Fast Garnet GBC/ml erprobt. Die besten Resultate liefert unabhängig von Gewebevorbehandlung und Organ hexazotiertes Neufuchsin.Im Vergleich zur Azofarbstoffreaktion mit Naphthol-AS-BI--d-N-acetylglucosaminid und hexazotiertem p-Rosanilin oder Neufuchsin oder tetrazotiertem BAXD liefert speziell die Azoindoxylmethode mit hexazotiertem Neufuchsin bessere oder identische Resultate. Die Indigogen-, Metallsalzund Tetrazoliumreaktion sind dem Azoindoxylverfahren meistens unterlegen; eine Ausnahme macht die Tetrazoliummethode mit BSPT.Beim Azoindoxylverfahren mit Hexazonium-p-rosanilin ist vorteilhaft, daß der Azoindoxylfarbstoff osmiert werden kann, in organischen Solventien und Kunstharzen weitgehend unlöslich ist und deshalb für die ultracytochemische Darstellung der -N-Acetylglucosaminidase in Frage kommt. Unter den übrigen Methoden ist dies nur noch mit der Tetrazoliumreaktion und BSPT der Fall; sein Formazan läßt sich ebenfalls osmieren.Mit hexazotiertem Neufuchsin zur Simultankupplung und 5-Br-3-Indolyl--d-glucosaminid als Substrat kann die -N-Acetylglucosaminidase nach Blockfixation in Form- oder Glutaraldehyd in den Lysosomen zahlreicher Rattenorgane und-gewebe einwandfrei nachgewiesen werden.

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Azoindoxyl methods for the investigation of hydrolasesIII. Histochemical studies of -d-N-acetylglucosaminidase

Summary The suitability of various azoindoxyl procedures for the light microscopical demonstration of -N-acetylglucosaminidase is described. The incubation media tried consist of 0.5 mg N-Acetyl-(5-bromindol-3-yl)--d-glucosaminide (5-Br-3-indolyl--d-N-acetylglucosaminide; 1 mg dissolved in 0.05 ml N,N-dimethylformamide) in 1 ml 0.1 M citric acid phosphate buffer, pH 4.5 or 5. 0.02 ml hexazotized p-rosaniline or new fuchsine/ml or tetrazotized BAXD or 0.5 mg Fast Blue B or Garnet GBC/ml were employed as a coupling reagent. Hexazotized new fuchsine yields the best results independent on the pretreatment of the tissue and the organ investigated followed by hexazonium-p-rosaniline.Compared with the azo dye method using naphthol AS-BI -d-N-acetyl-glucosaminide as a substrate and hexazotized p-rosaniline or new fuchsine or tetrazotized BAXD for simultaneous coupling especially the azoindoxyl technique with the new fuchsine is equivalent or superior. When the indolyl glucosaminide is used in the indigogenic, tetrazolium or metal precipitation method the results are mostly inferior with the exception of the tetrazolium reaction using BSPT.However, the main advantage of the azoindoxyl procedure is that at least the azoindoxyl dye deriving from hexazotized p-rosaniline can be osmificated and withstands treatment with organic solvents and resins. Therefore, the reaction product seems to be suitable for the electron microscopic demonstration of glucosaminidase. Among the other reaction principles this can reliably be achieved only with BSPT as a tetrazolium salt followed by osmification of its formazan.After fixation of blocks of tissue in form- or glutaraldehyde -d-N-acetylglucosaminidase can be localized with 5-Br-3-indoxyl--d-N-acetylglucosaminide as a substrate and hexazotized new fuchsine for simultaneous coupling in the lysosomes of many rat organs.

     

Cell multiplication and ultrastructure ofMicrasterias denticulata (Desmidiaceae) grown under salt stress

Cells ofMicrasterias denticulata Bréb. were kept in nutrient solution of high osmolality (salt stress) for four weeks. In a special cell multiplication test it was established that cell division is gradually inhibited at increasing salt concentrations and totally arrested at the highest concentration (26 mosm/kg). Recovery studies proved that even cells from the highest concentration range start dividing immediately after being placed in aqua bidest. thus indicating the full reversibility of the inhibiting effect. — Cells of the highest concentration range show marked ultrastructural changes. Besides an enormous accumulation of starch and oil bodies and a condensed appearance of the ground plasma, a reduction of mitochondria, ER and the Golgi-system is found. The most striking effect occurs on the vacuolar system which appears extremely reduced and condensed. The cell wall is thickened by the formation of an additional cell wall layer with a spongy electron microscopical appearance. Through the cell wall many droplets of a probably fat-like substance are excreted. — In summary, salt stress induces growth-inhibited akinete cells in the sense ofFritsch; these can be reactivated by decreasing the salt concentration. The salt-induced akinete state seems to be an ecological adaption to unfavourable conditions rather than a degeneration of the cells.Dedicated to Prof. DrLothar Geitler on the occasion of his 90th birthday.23. 12. 1988     

Endocrine cells in the human fetal small intestine

Summary In this report we describe the time of appearance and ultrastructural features of enteroendocrine cells (EECs) in the human fetal small intestine (SB) between 9 and 22 weeks gestation. Thirteen distinctive EECs were identified in fetal SB. Two of these, not found in normal adult SB, appeared within the stratified epithelium of the proximal SB at 9–10 weeks. They were arbitrarily termed primitive and precursor cells. As in all fetal EECs, the pale cytoplasm of the primitive cell contains a distinctive population of secretory granules (SGs). Primitive cell SGs average 200–330 nm; some have dense cores with lucent halos while others are filled with a homogeneous dense or flocculent material. The SGs of the precursor cells are larger, averaging up to 1 m in diameter and their contents vary in electron density. A third group of cells not described in normal adult SB was arbitrarily termed transitional cells. These have two populations of SGs; one resembles the SGs of the precursor cells, and the other resembles the SGs of some of the specific adult type EECs. Transitional EC, S, I and G cells are seen. In addition, mature appearing EC, S, G, I, L, D, and D₁ cells were identified by 12 weeks of gestation. The primitive, precursor, and transitional cells may represent sequential developmental precursors of adult type EECs.Supported by Research Grant AM-17537 from the National Institutes of Health, Besthesda, MarylandThe authors would like to thank Ms. Linda Barstein for her excellent technical assistance     

Callus induction and plant regeneration in Vetiveria zizanioides

Callus induction was obtained from basal parts of Vetiveria zizanioides Stapf. leaves cultured on Murashige and Skoog (MS) medium supplemented with 9.0 M 2,4-dichlorophenoxyacetic acid (2,4-d), 5.7 M indoleacetic acid (IAA) and 4.6 M kinetin. Calli were maintained on MS medium with the addition of 0.9 M 2,4-d and 2.3 M kinetin. Shoot formation was obtained from fast growing 14-day-old callus on the same basal medium supplemented with 0.9 M 2,4-d and 9.3 M kinetin. Embryo-like structures were observed. When transferred to basal medium, shoots readily developed roots. Fully developed regenerated plants were then successfully established in soil.