A fluorometric assay of ceramide glycanase with 4-methylumbelliferyl β-d-lactoside derivatives

4-Methylumbelliferyl 6-O-benzyl--d-lactoside (6Bn-MU-Lac) and some related compounds were synthesizedvia different selective reactions including phase-transfer glycosylation. Their suitability as substrates for a fluorometric assay of ceramide glycanase (CGase) was evaluated. Among others, the 6Bn-MU-Lac, which is resistant to exogalactosidase, was found to be a suitable substrate for routine assay of the CGase activity. For American leech CGase, theK _m value is 0.232 mM at pH 5. Abbreviations: CGase, ceramide glycanase; Gal, galactose; Glc, Glucose; Lac, lactose; MU, 4-methylumbelliferone; MU-Lac, 4-methylumbelliferyl -d-lactoside; bBn-Lac, 6-O-benzyl-lactose; 6Bn-MU-Lac, 4-methylumbelliferyl 6-Obenzyl--d-lactoside; 46Bd-MU-Lac, 4-methylumbelliferyl 4,6-O-benzylidene--d-lactoside; MU-Cel, 4-methylumbellifery -d-cellobioside; 46Bd-MU-Cel, 4-methylumbelliferyl 4,6-O-benzylidene--d-cellobioside; TLC, thin layer chromatography;¹H-NMR, proton nuclear magnetic resonance; GSL, glycosphingolipids; CSA, 10-camphorsulfonic acid. See Scheme 1 for chemical structures.     

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     

Protein kinase C (α, β, γ) in Pacinian corpuscle

Immunocytochemical demonstration of protein kinase C (PKC) subspecies (, , ) was carried out in Pacinian corpuscles of rat hind feet using monoclonal or polyclonal antibodies against each of these subspecies. The inner core cells and lamellae and the Schwann cell cytoplasm of the nerve fiber innervating the corpuscle were strongly positive for PKC -immunoreactivity (IR). In contrast, the axon terminal and the outer core did not display any positive -IR. Very weak PKC -IR was detected in the ultraterminal region of the axon terminal, while the trunk region showed no immunoreactivity. Very faint PKC -IR was found also in the lamellar cells located at the periphery of the inner core and the endoneurial fibroblasts in the intermediate layer. PKC -IR was not detected in any part of the corpuscle. The strong PKC -IR in the inner core and the presence of absence of PKC -, -, and -IR in the axon terminal are discussed from the point of view of the functional aspects of each part.     

Hexagonally ordered “rosettes” of particles in the plasma membrane ofMicrasterias denticulata Bréb. and their significance for microfibril formation and orientation

Summary Cells ofMicrasterias denticulata Bréb. at the stage of secondary wall formation have been studied by freeze-etching. It was found that the plasma membrane exhibits oval areas in which arrays of membrane particles occur. These particles form rosettes which are arranged in a hexagonally ordered lattice with a center to center spacing of 25 nm. Nearly the same periodicities can be found between microfibrils. It is concluded that the rosettes probably together with the thickened area of the plasma membrane below them represent the apparatus for the production and orientation of microfibrils. The hypothesis suggesting the incorporation of membrane templates functional in microfibril formation, originally advanced byKiermayer andDobberstein (1973) has received further support.     

Enzymic synthesis of the trisaccharide core region of the carbohydrate chain ofN-glycoprotein

Transmannosylation from mannotriose (Man1-4Man1-4Man) to the 4-position at the nonreducing end N-acetylglucosaminyl residue ofN,N-diacetylchitobiose was regioselectively induced through the use of -d-mannanase fromAspergillus niger. The enzyme formed the trisaccharide Man1-4GlcNAc1-4GlcNAc (3.7% of the enzyme-catalysed net decrease ofN,N-diacetylchitobiose) from mannotriose as a donor andN,N-diacetylchitobiose as an acceptor. Mannobiose (Man1-4Man) was also shown to be useful as a donor substrate for the desired trisaccharide synthesis.Abbreviations Man d-mannose - (M _n) (n=1–5) -linkedn-mer of mannose - GlcNAc₂ 2-acetamido-2-deoxy--d-glucopyranosyl-(1–4)-2-acetamido-2-deoxy-d-glucose     

Electron microscopic studies on the adenohypophysis of the White-Crowned sparrow,Zonotrichia leucophrys gambelii

Summary The pars distalis of the adenohypophysis of normal (78), thyroideotomized (6), adrenalectomized (6), and castrated (14) White-crowned Sparrows were observed with the electron microscope. Six types of glandular cells were identified and the ultrastructural characteristics of each have been described. To each has been assigned tentatively an endocrine function.STH cells are characterized by the presence of large, dense secretory granules ranging from 220–280 m, a poorly developed endoplasmic reticulum, and a fragmented Golgi apparatus; they occur only in the caudal lobe. They show no remarkable changes after adrenalectomy, castration, and thyroidectomy.Prolactin cells, whose identity is suggested by their responses to photostimulation and surgical experiments, are characterized by large, polymorphic, dense secretory granules; they have been found mainly in the cephalic lobe.ACTH cells, whose function is confirmed by their cytological responses to adrenalectomy, have a peculiar type of secretory granule (220 m) with high and low phases of electron density. They occur exclusively in the cephalic lobe and are transformed, after adrenalectomy to large, vacuolated adrenalectomy cells.TSH cells are so designated by their response to thyroidectomy. After thyroidectomy, they lose their specific fine secretory granules and are transformed into large, vacuolated thyroidectomy cells. We have found TSH cells and thyroidectomy cells only in the cephalic lobe.Two types considered to be gonadotropic cells from their responses to gonadectomy, occur in both the cephalic and caudal lobes. One of them contains spherical, dense secretory granules (180–220 m), prominent rough endoplasmic reticulum and a well developed Golgi apparatus; the other type contains dense secretory granules of variable size (150–350 m), a less extensively developed Golgi apparatus, and sac-like endoplasmic reticulum. Both types of gonadotropic cells show extreme enlargement and vacuolization after castration. However, they retain differences in appearance in the structure of cytoplasmic organelles and vacuolization.Dedicated to Professor Dr. H. Grau in honor of his 70th birthday.The investigation reported herein was supported by a research grant (HE 07240 NEUA) from the National Institutes of Health to Professor Vitums, by a research grant (5R01 NB 06187) from the National Institutes of Health to Professor Farner, and by a scientific research grant (No. 91049) from the Ministry of Education of Japan to Professor Mikami. The authors wish to thank Professor James R. King for his assistance in obtaining and maintaining the birds, and for his helpful advice concerning the experiments.     

Synthesis of methyl α- and β-N-dansyl-d-galactosaminides,probes for the combining sites ofN-acetyl-d-galactosamine-specific lectins

The synthesis of the methyl - and -N-dansyl-d-galactosaminides is described using methyl ,-2-azido-2-deoxy-d-galactopyranoside as starting material. This was reduced to the corresponding methyl ,-2-amino-2-deoxy-d-galactopyranoside and then treated with dansyl chloride to yield a mixture of methyl ,-N-dansyl-d-galactosaminides which was separated into individual anomeric forms by flash chromatography on silica gel. Methyl -N-dansyl-d-galactosaminide was used as a fluorescent indicator ligand in continuous substitution titrations to determine the association constants of nonchromophoric carbohydrates with theN-acetyl-d-galactosamine specific lectin fromErythrina corallodendron.Abbreviations ECorL Erythrina corallodendron lectin - MeGalNDns methyl 2-deoxy-2-(5-dimethylamino-1-naphthalenesulfamido)--d-galactopyranoside - MeGalNDns methyl 2-deoxy-2-(5-dimethylamino-1-naphthalenesulfamido)--d-galactopyranoside Dedicated to Hilde De Boeck (1958–1991).     

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.     

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.     

Properties of β-glucosidase purified from Aspergillus niger mutants USDB 0827 and USDB 0828

Summary Two extracellular -glucosidases (EC 3.2.1.21) were isolated from Aspergillus niger USDB 0827 and A. niger USDB 0828, and their physical and kinetic properties studied. Both enzymes were very similar in terms of molecular size (230000 Da), pH optimum (pH 4.6), temperature optimum (65° C), stability at high temperatures and substrate preferences. They were capable of hydrolysing -linked disaccharides, phenyl -d-glucoside, p-nitrophenyl -d-glucoside (PNPG), o-nitrophenyl -d-glucoside, salicin and methyl -d-glucoside but lacked activity towards -linked disaccharides, a range of p-nitrophenyl monoglycosides and p-nitrophenyl diglycosides. Both -glucosidases were better at hydrolysing cellobiose than cellotriose, cellotetraose or cellopentaose. For both enzymes, glucose showed competitive inhibition with PNPG as substrate but had no effect with cellobiose. However, the two -glucosidases differed in inhibition by glucono-1,5-lactone and affinity for cellobiose. -Glucosidase from A. niger USDB 0827 also gave lower specific activity, and was more susceptible to metal ions (Ag⁺, Fe²⁺ and Fe³⁺) inhibition than that of A. niger USDB 0828. Correspondence to: Y. K. Hoh     

Photosynthese und Photomorphogenese bei Farnvorkeimen vonDryopteris filix-mas

Summary Under conditions of identical rate of photosynthesis (measured by dry weight increase under steady state conditions) growth and differentiation of the gametophytes of ferns (e.g.Dryopteris filix-mas) are completely different in red and blue light. In the blue light normal growth and morphogenesis take place and normal two or three-dimensional prothallia are formed (Fig. 3). In the red, however, the prothallia look very similar to those growing in complete darkness: they grow as one-dimensional filaments (Fig. 1).It has been shown in this paper that photosynthesis, which is important as a source of organic material and free energy, has no influence at all on morphogenesis. Morphogenesis, i.e. the formation of normal prothallia instead of filaments, is controlled by a photoreactive system which depends on blue light of suitable intensity and which is not related to photosynthesis as such. If no blue light is present no morphogenesis occurs in spite of high photosynthetic activity.In our opinion theprimary products of photosynthesis are the same in all wavelengths. But now the photomorphogenic light reaction which depends on blue light apparently directs the flow of metabolites. In this way even the same initial products of CO₂ fixation may lead subsequently to rather different photosynthetic products and consequently to the very great difference between prothallia growing with or without blue light.The addition of sucrose has practically no influence on growth and morphogenesis under our conditions. On the basis of our results we cannot agree with the general conclusions drawn byMiller andMiller (1961) who regard photosynthesis as a photomorphogenetic system in these gametophytes of ferns.Mit 6 Textabbildungen.Herrn Professor Dr.E. G. Pringsheim in Verehrung zum 80. Geburtstag.     

On T-tubule openings at the sarcolemma of white fast-twitch muscle fibres in fish and frog

Summary In white muscle fibres of a teleost fish T-tubule openings may occur regularly at all Z-disc levels between adjacent peripheral myofibrils, the T-tubule openings thus occurring at a density of ca. 0.9 m^-2.In frog white fibres, T-tubule openings are infrequently seen in material fixed like the fish material. In material prepared according to the albumin method of Gray (1975, 1976 a, b) which renders the muscle fibres swollen, straight tubules or sometimes chains of vesicles instead are seen opening at the sarcolemmal surface. Such tubules occur at a higher density than expected from experiments with local activation of contraction. Lability and dynamics within the T-system normally and during fixation are discussed.     

Relation of nitrogen fertilizers and manure to vascular wilt of egyptian cotton caused by four fusaria species and strains

Summary Four different species and strains ofFusarium, namely,F. oxysporum f.vasinfectum (Atk.)Snyder &Hansen,F. solani (Mart.)App. &Wr.,F. vasinfectum (Atk.) f₁ Wr. strainFahmy, andF. vasinfectum Atk. are responsible for cotton wilt in U.A.R. Pathogenicity experiments have shown that the four experimentalFusaria possess varying degrees of pathogenicity towards two susceptible cotton varieties, namely, Karnak and Bahtim 190. On the other hand, Ashmouni cotton proved resistant to the fourFusaria. Variability of pathogenicity of the experimentalFusaria is not only related to host cotton plant or parasite, but also to nitrogenous fertilizer or manurial treatment of the soil. Resistance of Ashmouni cotton was not broken in presence of sodium nitrate, ammonium nitrate or stable manure added to soil inoculated with any of the fourFusaria. The application of nitrogen fertilizers or manure resulted on the whole, in increased wilt-disease incidence of the susceptible cotton varieties. Stable manure enhanced predisposition to vascular-wilt more than other nitrogen treatments, especially in presence of the virulentF. oxysporum f.vasinfectum or both strains ofF. vasinfectum Atk. Ammonium nitrate induced the sudden appearance of mottling characteristic of vascular-wilt of cotton when Bahtim 190 plants were raised in nitrogen-fertilized soil inoculated withF. solani (Mart.)App. &Wr. At the same time, the raised affected plants showed increased growth-vigour and enhanced maturity of their leaves.     

Developmental aspects of long-term callus culture ofVicia faba L.

Summary Vicia faba callus line (VFS 1), isolated from expiants of immature embryo, grew satisfactorily onMurashige andSkoog complete medium with 1.38 M 2,4-D, or with 0.92 M 2,4-D to which 1.0 M kinetin was added. It also grew well on the B 5 modified medium containing 2.3 M 2,4-D and 25.0 M kinetin. On the last of these media the cultures grew more uniformly and without necrosis. They also showed diminishing variation in polyploidy in favour of diploids and corresponding aneuploids (hypodiploids).After being cultured for nearly three years on MS containing 1.38 M 2,4-D, 8–33% of cultures of VFS 1 were able to regenerate roots when transferred to either MS half strength with 5.37 M NAA, or to a medium without 2,4-D, or else to media with the addition of kinetin only (in various concentrations).     

Über die Malat-Dehydrogenase-und Lactat-Dehydrogenase-Aktivität L-Äpfelsäure-abbauender Bakterien

Zusammenfassung Bei 6 verschiedenen Arten L-Äpfelsäure-abbauender Bakterien werden Untersuchungen über die Malat-Dehydrogenase- und Lactat-Dehydrogenase-Aktivität vorgenommen. Die heterofermentativen Arten (Leuconostoc r, La Louvière, Pf-1) besitzen eine ausgeprägte Malat-Dehydrogenase-Aktivität, während die homofermentativen nur eine schwache (L. QT 1-6) bzw. keine meßbare Aktivität (L. casei, L. plantarum [arabinosus 17-5]) aufweisen. Alle Stämme sind in hohem Maße Lactat-Dehydrogenase-aktiv.Die Synthese der Malat-Dehydrogenase ist induktiv; L-Äpfelsäure als Substrat verstärkt eine vorhandene schwache Aktivität. Beweisend hierfür ist die starke Abnahme der Umsatzzahlen für die L-Äpfelsäure-Dehydrierung bzw. die Oxalessigsäure-Hydrierung nach erfolgter Entwöhnung der Bakterien von Äpfelsäure, sowie der größere Zahlenwert der Michaelis-Konstanten. Auch die Synthese der Lactat-Dehydrogenase wird von L-Äpfelsäure induziert. Insofern dürfte die Synthese dieses Enzyms mit derjenigen der Malat-Dehydrogenase gekoppelt sein.Die Lactat-Dehydrogenase von Leuconostoc r und La Louvière läßt sich wie die tierische Lactat-Dehydrogenase durch Sulfit hemmen; die Empfindlichkeit gegenüber Sulfit ist etwa doppelt so hoch wie beim tierischen Enzym. Die Enthemmung kann außer mit Lactat auch mit d(+)- und L(-)-Malat erfolgen; Malat ist jedoch nur in höherer Konzentration wirksam. Zur Hemmung der bakteriellen Malat-Dehydrogenase sind mehrfach höhere Sulfit-Konzentrationen nötig als beim tierischen Enzym. Außer d(+)-Malat hat auch Lactat enthemmende Wirkung.

---

The malate dehydrogenase and lactate dehydrogenase activity of bacteria, decomposing L-malic acid

Summary The malate dehydrogenase and lactate dehydrogenase activities of six strains of bacteria, decomposing L-malic acid, were investigated. The heterofermentative strains (Leuconostoc r, La Louvière, Pf-1) possess a distinct malate dehydrogenase activity, while the homofermentative strains show only a weak (L. QT 1-6) respectively no measurable activity (L. casei, L. plantarum [arabinosus 17-5]). All strains possess a high lactate dehydrogenase activity.The synthesis of the malate dehydrogenase is inductive. The presence of L-malic acid intensifies a weak activity. This is shown by the strong decline of the turnover rates for the dehydrogenation of L-malic acid respectively the hydrogenation of oxaloacetic acid after deadaptation of the bacteria from malic acid, and by the greater values of the Michaelis' constants. Also the synthesis of the lactate dehydrogenase is induced by L-malic acid. Therefore the synthesis of this enzyme appears to be coupled with the synthesis of the malate dehydrogenase.The lactate dehydrogenase of Leuconostoc r and La Louvière can be inhibited by sulphite like the animal lactate dehydrogenase; the sensibility to sulphite is about the twofold of the animal enzyme. The reactivation can be achieved with lactate, or d(+)-and L(-)-malate; malate is effective only at higher concentrations. For the inhibition of the bacterial malate dehydrogenase manifold higher sulphiteion concentrations are needed than for the animal enzyme. Besides d(+)-malate also lactate has a reactivating effect.

---

---

III. und letzte Mitteilung zum Problem des biologischen Säureabbaues. Über adaptive und konstitutive fermentchemische Eigenschaften L-Äpfelsäure-abbauender Bakterien.     

Effect of culture medium ions on chromate reduction by resting cells of Agrobacterium radiobacter

The influence of some ions in pre-growth culture medium on chromate reduction by resting cells of Agrobacterium radiobacter strain EPS-916 was investigated. The reduction was dependent on the Fe²⁺ content of the culture medium: the higher the iron content, the lower the reduction rate. The cells showed maximum chromate reduction when pre-grown in the presence of 0.243 m Mg²⁺, 20 m Ca²⁺ and 3.6 m Mn²⁺. Chromate reduction was not affected by the addition of MgCl₂, CdCl₂, ZnCl₂, MnCl₂, Na₂SO₄ (1000 m), and Na₂MoO₄ (100 m) to the activity assays. However, activity was inhibited by the presence of Na₂SO₄ (10 mm), Na₂MoO₄ (200 m) and ferric citrate.     

Tissue culture and micropropagation of Cuphea ericoides,a potential source of medium-chain fatty acids

Plant rgeneration occurred on leaf-and stem-derived callus of Cuphea ericoides Cham. & Schlechtd obtained in Murashige and Skoog medium supplemented with auxins [indole-3-acetic acid (IAA), -naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-d)] plus cytokinins [6-benzyladenine (BA) or kinetin]. These calluses were subcultured and showed vigorous growth. When subcultured on medium containing 2.22 or 4.44 M BA, the calluses showed profuse regeneration of shoots whereas those subcultured on medium supplemented with 2.69 M NAA or 0.226 M 2,4-d produced numerous roots. Isolated shoots rooted on Murashige and Skoog medium lacking growth regulators or containing 0.54 M NAA or 0.49 M indole-3-butyric acid (IBA). Plantlets were acclimatized to greenhouse conditions.Abbreviations BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige & Skoog medium - NAA 1--naphthaleneacetic acid