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.     

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.     

Die Lebensformen der Laubmoose

Zusammenfassung Im Gegensatz zu dem morphologischen Begriff Wuchsform wird der ökologische Begriff Lebensform definiert als der Habitus einer Pflanze in Einklang mit ihren Lebensbedingungen. Es werden 10 Lebensformen der Laubmoose unterschieden, ihr Anteil an verschiedenen Pflanzenformationen und Moosgesellschaften dargelegt und ihre Abhängigkeit von den ökologischen Bedingungen erörtert.

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Summary In opposition to the morphological term growth-form the ecological term life-form is defined as the organization of a plant in correspondence with its life conditions. Ten life-forms of mosses are discerned, their part within various plant-formations and moss-associations is explained, and their dependence on ecological conditions is discussed.

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Herrn Prof. Dr. Carl Troll in herzlicher Verehrung zum 70. Geburtstag gewidmet.     

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     

Synapses in the cerebral ganglion of adult Ciona intestinalis

Summary Electron microscopy of the synaptic morphology of synapses in the cerebral ganglion of the adult ascidian (sea squirt) Ciona intestinalis reveals that the synapses are restricted to the central neuropil of the ganglion. Many of the synapses show a polarity of structure such that pre and post synaptic parts can be identified. The vesicles in the presynaptic bag are of two main diameters 80 and 30 nm respectively. The large vesicles have electron dense contents that vary both in their capacity and dimensions.The pre and postsynaptic membranes are more electron dense than the surrounding membranes, but they are only slightly thicker. Both the pre and post synaptic membranes have electron dense dots some 10 nm in diameter associated with their cytoplasmic surfaces. Sometimes the presynaptic membrane has larger peg-like projections between the vesicles. Associated with the post synaptic membrane are tubules some 10 nm in diameter. These tubules may be the dots cut obliquely.The synaptic cleft material is more electron dense than the surrounding intercellular material, and in it there is a dense line made up of granules about 3–5 nm in diameter. This dense line is usually mid way between the pre and post synaptic membranes, but may be nearer the postsynaptic membrane.No tight junctions between adjacent nerve process profiles have been observed.I wish to thank Professors J. Z. Young, F. R. S. and E. G. Gray for much advice and encouragement, also Dr. R. Bellairs for the use of electron microscope facilities and Mr. R. Moss and Mrs. J. Hamilton for skillful technical assistance.     

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.     

Über die Verwendung der reversibel polymeren Metachromasie von Pseudoisocyanin zur Gewebsfärbung

Zusammenfassung 1. Pseudoisocyanin gibt mit den dicht gelagerten elektronegativen Gruppen von Mukopolysacchariden in Geweben und Lösungen, wie auch mit synthetischen Produkten mit linear angeordneten elektronegativen Gruppen in Lösung wie z. B. Polyäthylensulfosäuren eine metachromatische Reaktion mit der charakteristischen langwelligen Bande (vgl.Scheibe u.Schauer 1958). Die elektronegativen Gruppen binden die Farbstoffmoleküle elektrostatisch und bilden die Gruppierung des reversiblen Polymerisates.2. Die metachromatische Reaktion mit der reversibel polymeren Bande läßt sich in Gewebsschnitten deutlich demonstrieren. Das Farbstoffpolymerisat absorbiert in Lösung bei der gleichen Wellenlänge wie im Gewebe, wodurch die Gleichheit der Vorgänge im Gewebe und in Lösung bewiesen ist.3. Das Pseudoisocyanin erscheint für die Darstellung von Mukopolysacchariden besonders geeignet, da nach früheren Arbeiten (Scheibe 1938,Zimmermann u.Scheibe 1956) schon eine monomolekulare Schicht die reversibel polymere Bande und damit die Metachromasie beobachtbar macht. Ferner sind bei Betrachtung der mit Pseudoisocyanin gefärbten Schnitte im monochromatischen Licht bei der Wellenlänge der polymeren Absorption Spuren von Mukopolysacchariden noch deutlich zu erkennen, die bei Betrachtung im weißen Licht unauffällig bleiben.4. An Hand einiger Beispiele (Mastzellen, Knorpelgewebe, hyalinisiertes Bindegewebe) wird die Verwendungsmöglichkeit in der Histochemie gezeigt.

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Summary 1. Pseudoisocyanin interacts with densly positioned electronegative groups of mucopolysaccharides in tissues and in solutions in the same way as it interacts with linear positioned electronegative groups of synthetic products in solution (for instance polyaethylensulfoacids). The metachromasia, which is due to this reaction of pseudoisocyanin with mucopolysaccharides shows a characteristic wave-band 5727 Å (Scheibe undSchauer 1958). The dye is bound electrostatically by the electronegative groups in form of a reversible polymerisate.2. The metachromatic reaction with the reversible polymerisate has been demonstrated in tissue-sections. The polymerisate with the dyestuff is shown to adsorb light at the same wavelength in tissues as in solutions. This finding confirms the identity of the reaction in tissues and in solutions.3. Pseudoisocyanin seems to be especially suited for the detection of mucopolysaccharides, for even a monomolecular layer of dyestuff allows the observation of the reversible polymeric band and therefore shows metachromasia. Further, after staining with pseudoisocyanin even small trans of mucopolysac charides which are not visible in the white light can be demonstrated by means of monochromatic light at the wave-length of the polymer absorption.4. As shown by staining mastcells, cartilage-tissue, hyaliniced connectivetissue, pseudoisocyanin seems to be of use for appliance in histochemistry.

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Mit 4 Textabbildungen     

Die optischen Übertragungseigenschaften der Komplexaugen von Drosophila

Summary The transfer properties of the optical system in the arthropod compound eye are determined by the interommatidial angle , influencing the resolving power, and by the width of the visual fields of single ommatidia , influencing the response at high spatial frequencies of brightness distributions in the object space. The energy transfer/ receptor is proportional to ( )² and decreases with in-inreasing approximation of the perfect-imaging condition: gD 0; 0. However, a value > 0 has to be maintained in order to overcome the threshold of nervous excitation at a certain minimum-brightness level. Theoretical treatment yields /=0.62 to 0.88 as the corresponding optimum-imaging relation. The actual ratio can be derived from measurements of the optomotor reactions to the movement of periodic brightness patterns. The approximate value 0.76 is obtained from the fruitfly Drosophila with normal and mutant eye pigmentation. As a result, the parameters of this imaging system are found to be established in a way that enables optimum performance at sufficient illumination. An dieser Stelle möchte ich Dr. W. Reichardt für sein eingehendes Interesse und manche anregende Diskussion über die Sehvorgänge im Komplexauge meinen Dank sagen. Dr. K. Kirschfeld verdanke ich ebenfalls wertvolle Hinweise. Herrn E. Freiberg bin ich für die Anfertigung der Abbildungen sehr verbunden.     

Differential maturation of μ and δ opioid receptors in the chick embryonic brain

The developmental profiles of the binding of and opiate receptors agonists was investigated using the chick embryo brain. Binding of opioids was performed at embryonic days 5, 6, 15, 18, and 20 in the developing chick embryo brain. [³H]dihyromorphine was used as a ligand and with 5×10^–7 M levorphanol for non-specific binding, and [³H](d-Ala²-d-Leu⁵)-enkephalin was used as a with 5×10^–7 M (d-Ser-Gly-Phe-Leu-Thr)-enkephalin for non-specific binding. Crude membranes were prepared from whole brain at days, 5, 6 and cerebral hemispheres at days 15, 18, and 20 of embryonic age. Both and opiate receptors were present during early embryogenesis and as early as day 5. Analysis of binding sites revealed high and low affinity sites during early embryogenesis but only one site. By 18 days of embryonic age, only one site remained. This developmental change is interpreted as a transitory state of the receptor to the adult pattern. The presence of only one site is constant throughout embryonic age; it is high during early embryogenesis reaching a lower level by 18 days. The presence of a dual binding site pattern for the receptor in early embryogenesis is implicated to have a functional significance in the pluripotential role of the endogenous opioids in early development.     

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.     

Klassifizierung mariner,brackiger und limnischer Grundwasserbiotope

Zusammenfassung 1. Das Küstengrundwasser stellt einen Übergangsbereich zwischen limnischen und marin beeinflußten Grundwasserbiotopen dar.2. Im limnischen, brackigen und marinen Mesopsammal beziehungsweise Mesopsephal wirken dieselben ökologischen Hauptfaktoren: Lichtlosigkeit und Ausmaß der Kavernengeräumigkeit. Diese ökologische Gemeinsamkeit gibt Anlaß, eine Gliederung der limnischen Grundwasserbiotope (Husmann 1966) zu einer Typologie der Gesamtheit limnischer, brackiger und mariner Subterranbiotope zu erweitern.3. Zur Gliederung der limnischen Grundwässer wird die Beschaffenheit des grundwasserführenden Substrates jeweils mit der Art und Weise zönologischer Einflüsse aus Oberflächengewässern, oder mit dem Fehlen derartiger Kontakte, in Beziehung gesetzt. Dabei ergeben sich die in Abbildung 1 genannten Bezeichnungen limnischer unterirdischer Biotope.4. Für eine Typologie der marin beeinflußten Interstitialgewässer — Thalassopsammal, Thalassopsephal — wird die Salinität des Interstitialwassers hinzugezogen.5. Unter Berücksichtigung der Vorbehalte vonDen Hartog (1964) wird dem Venedig-System hierzu nur beschränkte Geeignetheit zuerkannt.6. Eine Heranziehung des Venedig-Systems beschränkt sich auf Grundwasserbiotope der Meeresküste mit besonders ausgeprägter Stabilität der Salinität. Ein Beispiel für eine derartige Besonderheit gibt Abbildung 1.7. Eine Kombination der vorgeschlagenen Bezeichnungen brackiger Interstitialgewässer mit dem Typologischen System der Brackwässer (Den Hartog 1964) erscheint nach Möglichkeit angebracht. Beispiel: Lagunäres (mixo-)oligohalines Thalassopsammal.

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Classification of marine, brackish and limnic groundwater biotopes

The oligohaline groundwater of marine beaches (Küstengrundwasser;Remane) represents an ecological zone of contact between limnic and marine groundwater biotopes. In the Küstengrundwasser and in all other brackish, marine and limnic interstitial waters there are two principal ecological factors: darkness and dimension of interstitial volume. These ecological conditions suggest a comprehensive classification of limnic, brackish and marine groundwater biotopes (except saline subterranean inland waters). Freshwater subterranean biotopes are classified in regard to the nature of the substrate containing groundwater, and the nature of contact with surface waters. The classification of subterranean biotopes influenced by marine conditions is based on the same factors plus salinity of the interstitial water. The Venice system for classification of brackish water is considered to be of limited value. In general, brackish subterranean waters should only be classified as: oligohaline, mesohaline or polyhaline thalassopsammal. The usefulness of the Venice system classification is limited to marine-influenced groundwater biotopes with an extremely stable salinity.

     

Design of superactive and selective integrin receptor antagonists containing the RGD sequence

Summary Integrins play a major role in cell-cell and cell-matrix interactions. The majority of the different types of integrins recognize the tripeptide sequence arginine-glycine-aspartic acid (RGD). To explore the spatial requirements of the pharmacophore for receptor selectivity and high activity, a new procedure, spatial screening, was used. The procedure is based on the experience that the conformation of small cyclic peptides is mainly determined by the chirality of the amino acids (and glycine or proline). For example, cyclic pentapeptides with one d and four l amino acids prefer a II'/ conformation. The sequence RGDFV was shifted around this spatial II'/ template by synthesis of five peptides in which one of the amino acids was used in d-configuration. It turned out that cyclo(-RGDfV-) is a selective inhibitor for the _v3 integrin, which is strongly expressed in cancer cells. Systematic variations with different turn mimetics, retro-inverso structures, modified peptide bonds and sugar amino acids are discussed.     

Interactions between anion exchange and other membrane proteins in rabbit kidney medullary collecting duct cells

Summary In separated outer medullary collecting duct (MCD) cells, the time course of binding of the fluorescent stilbene anion exchange inhibitor, DBDS (4,4-dibenzamido-2,2-stilbene disulfonate), to the MCD cell analog of band 3, the red blood cell (rbc) anion exchange protein, can be measured by the stopped-flow method and the reaction time constant, _DBDS, can be used to report on the conformational state of the band 3 analog. In order to validate the method we have now shown that the ID_50,DBDS,MCD (0.5±0.1 m) for the H₂-DIDS (4,4-diisothiocyano-2,2-dihydrostilbene disulfonate) inhibition of _DBDS is in agreement with the ID_50,Cl ^–,_MCD (0.94±0.07 m) for H₂-DIDS inhibition of MCD cell Cl^– flux, thus relating _DBDS directly to anion exchange. The specific cardiac glycoside cation transport inhibitor, ouabain, not only modulates DBDS binding kinetics, but also increases the time constant for Cl^– exchange by a factor of two, from _Cl=0.30±0.02 sec to 0.56±0.06 sec (30mm NaHCO₃). The ID_50,DBDS,MCD for the ouabain effect on DBDS binding kinetics is 0.003±0.001 m, so that binding is about an order of magnitude tighter than that for inhibition of rbc K⁺ flux (K _I,K ⁺,_rbc=0.017 m). These experiments indicate that the Na⁺,K^–-ATPase, required to maintain cation gradients across the MCD cell membrane, is close enough to the band 3 analog that conformational information can be exchanged. Cytochalasin E (CE), which binds to the spectrin/actin complex in rbc and other cells, modulates DBDS binding kinetics with a physiological ID_50,DBDS,MCD (0.076±0.005 m); 2 m CE also more than doubles the Cl^– exchange time constant from 0.20±0.04 sec to 0.50±0.08 sec (30mm NaHCO₃). These experiments indicate that conformational information can also be exchanged between the MCD cell band 3 analog and the MCD cell cytoskeleton.     

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     

Inhibition of tumor growth in mice treated with synthetic muramyl dipeptide

Summary Treatment with synthetic MDP inhibited growth of transplantable, chemically induced tumors in syngeneic mice. The tumor-inhibitory effect was dependent on the schedule of MDP administration.Growth of SC transplants of a nonmetastasizing, MC-induced fibrosarcoma, MC11, was inhibited by local treatment with 200 g and 1,000 g MDP given SC 5–7 weeks before challenge. Treatment with lower (10 g and 100 g) doses of MDP and shorter (1–4 weeks) time intervals was not effective. Single doses of MDP (10–1,000 g) 1–3 weeks after challenge had no effect.Growth of IV-inoculated, metastasizing AAT-induced hepatoma A was inhibited by IV injections of 20 g MDP given 1 and 2 days prior to the challenge. Significant increases in the survival of hepatoma-bearing mice were observed only after injections of MDP incorporated in multilamellar liposomes.Abbreviations MDP n-acetylmuramyl-l-alanyl-d-isoglutamine - B10 C57BL/10ScSnPh mice - MC 3-methylcholanthrene - ATT o-amino-azotoluene - PBS phosphate-buffered saline     

Plantlet regeneration from callus initiated from flower buds in the wild species Allium senescens var. minor

Callus regeneration was observed from flower buds of Allium senescens var. minor inoculated in BDS, MS or B5 medium supplemented with 4.4 M benzyladenine alone or in combination with 4.5 M 2,4-dichlorophenoxy-acetic acid (2,4-d), with 2,4-d and kinetin (4.5 M/4.6 M) or with 5.3 M naphthaleneacetic acid. Ovules enlarged initially but the embryogenic tissue degenerated as callus development progressed from the nectar regions of the petals. Shoot buds and leaf primordia developed from the meristematic protuberances that originated from the surface of the callus. BDS medium with 4.5 M 2,4-d and 13.3 M BA was most suitable for shoot multiplication. The regenerated shoots were rooted in respective liquid medium without any growth regulators and successfully transferred to soil with 90% survival rate.Abbreviations BA N⁶-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid     

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.

     

Flexibility on the karyotype evolution in bitterlings (Pisces, Cyprinidae)

In bitterlings (Acheilognathinae) C- and Ag-banding karyotypes of 6 species-subspecies collected in China and South Korea were analyzed. The chromosomal constitution of 2n=46 (4SM+42ST) in Rhodeus atremius fangi was quite different from that of 2n=48 (8M+20SM+20ST) in other species-subspecies in Rhodeus. It was concluded from the analysis of banded chromosomes that the increase in number of ST during the karyotype change from 2n=48 to 2n=46 was achieved by a series of pericentric inversions from 24 M-SM to 24 ST, and the decrease in the diploid number was caused by an additional tandem fusion of 4 ST chromosomes, forming a new ST pair in the 2n=46 karyotype. The karyotype of Tanakia koreensis, T. signifer, and Acheilognathus macropterus is 2n=48 (8M+20SM+20ST), 2n=48 (8M+20SM+14–16ST+4–6 A), 2n=44 (14M+16SM+14ST), respectively. In R. ocellatus ocellatus, T. koreensis, T. signifer and A. macropterus, karyotype changes from 2n=48 to 2n=44 due to centric fusion and inversion have also been estimated. It was suggested that C-banding heterochromatin was greatly concerned with the karyotype evolution in bitterlings.     

The rapid switch-off of nitrogenase activity in obligate methane-oxidizing bacteria

Nitrogenase activity in the obligate methaneoxidizing bacterium Methylococcus capsulatus (Bath) was added ammonia. This observation was extended to include other ammonia. This observation was extended to include other representative N₂-fixing species of methanotrophs. The ammonia switch-off of nitrogenase in M. capsulatus (Bath) was reversed on washing cells to remove excess ammonia, in the presence of chloramphenicol, suggesting that a form of covalent modification of nitrogenase may occur. Replacing the oxidizable substrate methanol with formaldehyde, formate, ethanol or hydrogen had no effect on nitrogenase switch-off. A number of potential nitrogen sources or intermediates of nitrogen metabolism such as glutamine, asparagine, glutamate and alanine when tested, did not effect switch-off. However, the rapid inhibition of nitrogenase activity of M. capsulatus (Bath) could be achieved by adding the uncoupler carbonylcyanide m-chlorophenylhydrazone or nitrite. The glutamine synthetase inhibitor methionine sulphoximine blocked the switch-off effect of ammonia, indicating that the metabolism of ammonia may be essential for switch-off to occur. Inhibitors of glutamate synthase did not alleviate the ammonia switch-off response. Methionine sulphoximine did not alleviate the rapid inhibition of nitrogenase by carbonylcyanide m-chlorophenylhydrazone indicating that the shortterm regulation of nitrogenase by uncouplers and ammonia proceed via different mechanisms.Abbreviations MSX methionine-DL-sulphoximine - DON 6-diazo-5-oxo-L-norleucine - GS glutamine synthetase - GOGAT glutamine 2-oxoglutarate aminotransferase (glutamate synthase) - CCCP carbonylcyanide m-chlorophenyl hydrazone