The Catalytic Transition State in ATP Synthase

The catalytic transition state of ATP synthase has been characterized and modeled by combined use of (1) Mg-ADP–fluoroaluminate, Mg-ADP–fluoroscandium, and corresponding Mg-IDP–fluorometals as transition-state analogs; (2) fluorescence signals of -Trp331 and -Trp148 as optical probes to assess formation of the transition state; (3) mutations of critical catalytic residues to determine side-chain ligands required to stabilize the transition state. Rate acceleration by positive catalytic site cooperativity is explained as due to mobility of -Arg376, acting as an arginine finger residue, which interacts with nucleotide specifically at the transition state step of catalysis, not with Mg-ATP- or Mg-ADP-bound ground states. We speculate that formation and collapse of the transition state may engender catalytic site / subunit-interface conformational movement, which is linked to -subunit rotation.     

Reassessment of the acceptor specificity and general properties of the Lewis blood-group gene associated α-3/4-fucosyltransferase purified from human milk

The acceptor specificity and general properties of a Lewis blood-group gene associated -3/4-L-fucosyltransferase isolated from human milk have been examined at the penultimate purification stage involving affinity chromatography on GDP-hexanolamine Sepharose, and after a subsequent gel filtration step on Sephacryl S-200. Both preparations transferred fucose to theO-4 position ofN-acetylglucosamine in Type 1 (Gal1-3GlcNAc-R) acceptors and theO-3 position of glucose in lactose-based (Gal1-4Glc) oligosaccharides, and both used Type 1 sialylated compounds when the terminalN-acetylneuraminic acid was present in -2,3 linkage. The striking difference between the two preparations was in their reactivity with Type 2 (Gal1-4GlcNAc-R) chains; after Sephacryl S-200 chromatography the apparentK _M values for the -3/4- preparation with unsubstituted low-molecular-weight Type 2 oligosaccharides were considerably increased. Substitution of the terminal galactose with sialic acid in -2,3 linkage decreased theK _M values for low-molecular-weight oligosaccharides but no detectable incorporation of fucose was observed intoN-acetyllactosamine end-groups of glycoproteins withN-linked oligosaccharide chains, irrespective of the presence of sialic acid in the terminal sequences.Deceased 25 June 1991.     

The histochemistry of glycoconjugates in the colonic epithelium of the chicken

Summary In the colonic epithelium of the chicken, glycoconjugates have been studied by means of selected histochemical methods of light and electron microscopy. According to the results obtained, most of the colonic goblet cells contained acidic and neutral glycoconjugates with sulphate and vicinal diol groupings, -D-mannose and -D-glucose residues and sialic acid-galactose dimers. These goblet cells were found to undergo changes in histochemical reactivity during upward migration along the crypts; -D-mannose and -D-glucose residues and terminal sialic acidgalactose dimers increased in amount. The striated border of the colonic columnar cells has, likewise, been found to contain such glycoconjugates as were similar in reactivity to those of the goblet cells. The histophysiological significances of glycoconjugates involved in the chicken colonic epithelium have been discussed with special reference to the functional activities of the carbohydrates.     

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     

Polyvalent GalNAcα1→Ser/Thr (Tn) and Galβ1→3GalNAcα1→Ser/Thr (T <Subscript>α</Subscript>) as the most potent recognition factors involved in <Emphasis Type="Italic">Maclura pomifera</Emphasis> agglutinin–glycan interactions

Summary The agglutinin isolated from the seeds of Maclura pomifera (MPA) recognizes a mucin-type disaccharide sequence, Gal13GalNAc (T) on a human erythrocyte membrane. We have utilized the enzyme-linked lectinosorbent assay (ELLSA) and inhibition assay to more systematically analyze the carbohydrate specificity of MPA with glyco-recognition factors and mammalian Gal/GalNAc structural units in lectin–glycoform interactions. From the results, it is concluded that the high densities of polyvalent GalNAc1Ser/Thr (Tn) and Gal13GalNAc1Ser/Thr (T) glycotopes in macromolecules are the most critical factors for MPA binding, being on a nanogram basis 2.0 × 10⁵, 4.6 × 10⁴ and 3.9 × 10⁴ more active than monovalent Gal, monomeric T and Tn glycotope, respectively. Other carbohydrate structural units in mammalian glycoconjugates, such as human blood group Sd (a⁺) related disaccharide (GalNAc14Gal) and P^k/P₁ active disaccharide (Gal14Gal) were inactive. These results demonstrate that the configurations of carbon-4 and carbon-2 are essential for MPA binding and establish the importance of affinity enhancement by high-density polyvalencies of Tn/T glycotopes in MPA–glycan interactions. The overall binding profile of MPA can be defined in decreasing order as high density of polyvalent Tn/T (M.W. > 4.0 × 10⁴) >> Tn-containing glycopeptides (M.W. < 3.0 × 10³) > monomeric T/Tn and P (GalNAc13Gal) > GalNAc > Gal >> Man, LAra, DFuc and Glc (inactive). Our findings should aid in the selection of this lectin for elucidating functions of carbohydrate chains in life processes and for applications in the biomedical sciences.     

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.     

A new histochemical method for the selective periodate oxidation of total tissue sialic acids

Summary Evaluation of the intensity of the periodic acid—Schiff (PAS) staining produced following oxidation for 1 h at 4°C with 0.4mm periodic acid in approximately 1m hydrochloric acid indicated that this reagent completely oxidized all available sialic acid residues of either the sialo- or sialosulphoglycoproteins of human and rat colon or the sialoglycoproteins of rat sublingual gland. These conditions produced no visible Schiff staining of either neutral macromolecules orvicinal diols located on hexose, 6-deoxyhexose orN-acetylhexosamine residues (neutral sugars) of sialo- and sialosulphoglycoproteins. Furthermore, there was no extraction of epithelial glycoproteins or de-O-acylation of side chain substituted sialic acid residues. These data demonstrate that 0.4mm periodic acid in approximately 1m hydrochloric acid can be used as a specific reagent for the selective visualization of sialic acids in the PAS procedure.Studies of the mechanism of the oxidation of neutral sugars with 0.4mm periodic acid in approximately 1m hydrochloric acid indicated that their lack of PAS reactivity was not due to the production of Schiff unreactive hemiacetals or hemialdals. It is suggested that the selectivity of 0.4mm periodic acid in approximately 1m hydrochloric acid is a result of an increase in the rate of the oxidation of the sialic acid residues together with a decrease in the rate of oxidation of neutral sugars.     

The mode of secretion of α-amylase in barley aleurone layers

The involvement of the endomenbrane system of barley (Hordeum vulgare L.) aleurone cells in the secretion of gibberellin-induced hydrolases has been investigated at the biochemical level. Our results show that at least 40–60% of the -amylase activity in homogenates of aleurone layers occurs in a membrane-bound, latent form. The latent -amylase can be assayed quantitatively following disruption of membranes by treatment with Triton X-100, ethanol, sonication, or osmotic shock and shear. The association of -amylase with the membrane is not an artifact arising from homogenization of the tissue, and acid protease is also enriched in the same subcellular fraction as the -amylase. The membrane fraction with which the -amylase is associated has many properties of the endoplasmic reticulum (ER). When membranebound -amylase is prepared in buffers containing 3 mM MgCl₂ two fractions from a sucrose step gradient contain most of the -amylase activity. These fractions are enriched in the ER marker enzyme, NADH-dependent cytochrome-c reductase, and show densities characteristic of smooth and rough ER during subsequent purification on continuous gradients. In step gradients prepared with ethylenediaminete-traacetic-acid-treated membranes, -amylase activity is contained primarily in one fraction having the density of smooth ER. Electron microscopy of the purified fractions is consistent with -amylase being associated with smooth and rough ER. However, it has not been ruled out that the enzyme is also associated with plasma membrane, Golgi membranes, or tonoplast. Examination of the isoenzyme patterns of secreted, of total-homogenate and of membrane-associated -amylases, as well as the results from pulsechase experiments using L-[³H]leucine for labeling of -amylase, are all consistent with the hypothesis that membrane-associated -amylase is an intermediate in the secretory process.Abbreviations CNTPE N-carbobenzoxy-L-tyrosine p-nitrophenylester - Cyt oxidase Cytochrome oxidase - ER endoplasmic reticulum - EDTA ethylenediaminetetraacetic acid - GA₃ gibberellic acid - IDPase inosine diphosphatase - K⁺-ATPase pH 6.5 K⁺-stimulated adenosine triphosphatase - MES 2-(N-morpholino)ethanesulfonic acid - MOPS 3-(N-morpholino)propanesulfonic acid - NADH: Cyt c reductase cyanide-insensitive NADH-linked cytochrome-c reductase - RER rough endoplasmic reticulum - Tris tris-(hydroxymethyl)-aminomethane     

Assessing glycosidic linkage flexibility: Conformational analysis of the repeating trisaccharide unit of Aeromonas salmonicida

Summary A detailed conformational analysis was performed for the synthetic branched trisaccharide -d-Man-NAc-(14)-[-d-Glc-(13)]-l-Rha 1 which represents the repeating unit of the O-antigenic polysaccharide of Aeromonas salmonicida. The study was based on 26 experimental NOE curves from 1D transient NOE experiments, employing Gaussian-shaped inversion pulses at 600 MHz. Eight of the NOE curves were interglycosidic and thus useful for an analysis of glycosidic linkage orientations. Metropolis Monte Carlo (MMC) simulations and minimum-energy calculations with the program GEGOP were used to obtain theoretical NOE curves which were compared to the experimental ones. MMC simulations with different temperature parameters of 310, 600, 900 and 2000 K allowed identification of NOEs which are sensitive towards different conformation distributions-not only different conformations-at both glycosidic linkages in 1. A comparison of trisaccharide 1 with the constituent disaccharides -d-ManNAc-(14)-l-Rha 2 and -d-Glc-(13)-l-Rha 3 revealed effects of branching on glycosidic linkage flexibility. A quantitative evaluation was facilitated by the introduction of entropy-related flexibility parameters. Our study indicates a notable restriction of flexibility, especially at the (13) linkage in 1. Although overall flexibility in 1 is reduced as compared to the constituent disaccharides 2 and 3, it cannot be neglected altogether. In summary, combined transient NOE experiments and MMC simulations provide a simple approach to analyse glycosidic linkage flexibility.     

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.     

Autoradiographische Untersuchungen zum Eiweiss- und Melanin-Stoffwechsel der verschiedenen Zellarten des Melanoms der Maus (Typ Harding-Passey)

Zusammenfassung Mäusen mit einem transplantierbarem Melanom, Typ Harding-Passey, wurde H-3-markiertes Dl-DOPA-, -T₂, Dl-DOPA-2,5,6-T₃, Dl-Prolin-2-T und L-Ty-rosin-3-T' injiziert und die H-3-Inkorporation in verschiedenen Zellarten des Melanom autoradiographisch zu verschiedenen Zeiten untersucht.H-3-DOPA wird selektiv in Melanin eingebaut. Dieses H-3-Melanin findet sich 1,5–24 Std nach Gabe des H-3-DOPA in Bindung an feine Melaningranula im Cytoplasma melaninarmer Melanocyten. Macrophagen dagegen enthalten zu dieser Zeit kaum H-3-Melanin. Nach 5 Tagen aber ist das H-3-Melanin fast ausschließlich in den Macrophagen nachzuweisen. Die Menge des synthetisierten H-3-Melanin war unabhängig davon, ob DOPA-, -T₂ oder DOPA-2,5,6-T₃ als H-3-Melaninvorstufe diente.Der Eiweißstoffwechsel wurde mit H-3-Prolin und H-3-Tyrosin untersucht. Die Eiweißneubildung in Melanoblasten und jugendlichen Melanocyten lag in der gleichen Größenordnung wie die der Leberparenchymzellen, während sie in Melaninspeicherzellen etwa zehnmal geringer war. Wurden die Versuchstiere erst 7 Tage nach Injektion der H-3-Aminosäure getötet, so war das H-3-Eiweiß aller Zellen zu 70–90% bereits wieder abgebaut, nur bei den Macrophagen stieg der Gehalt an H-3-Eiweiß auf das Doppelte an. Dies wird durch Phagocytose H-3-markierter Eiweiß-Melanin-Granula erklärt.Die H-3-Markierung des Tyrosin-3-T wurde nur zu einem autoradiographisch im Vergleich zur H-3-Markierung des Eiweißes nicht mehr faßbaren Bruchteil in Melanin eingebaut.Die Arbeit wurde durch Mittel der Deutschen Forschungsgemeinschaft und des Bundesministeriums für Atomkernenergie unterstützt.     

Amino acid activation and polymerization at modular multienzymes in nonribosomal peptide biosynthesis

Summary The biosynthesis of microbial bioactive peptides is accomplished nonribosomally by large multifunctional enzymes consisting of linearly arranged building blocks of 1,000–1,500 amino acid residues. Each of these units acts as an independent enzyme which catalyzes the selection, activation, and in some cases modification (epimerization, N-methylation) of its cognate amino acid, as well as the elongation of the peptide product. The specific linkage of amino acid activating modules upon such polyenzymes defines the sequence of the peptide product. A series of functional domains could be identified upon an amino acid activating module which are involved in the sequential reactions in nonribosomal peptide biosynthesis.Abbrevations aaRS aminoacyl tRNA synthetase - GS1 gramicidin S synthetase 1 (phenylalanine racemase) - GS2 gramicidin S synthetase 2 - TY1 and 2 tyrocidine synthetase 1 and 2 - ACV [-(l--aminoadipyl)-l-cysteinyl-d-valine] - FITC fluorescein 5-isothiocyanate - FAB-, ESI-MS fast atom bombardment-, electrospray ionization-mass spectrometry - Pan 4-phosphopantetheine - NMR nuclear magnetic resonance - ACP acyl carrier protein - SAM S-adenosyl-l-methionine - CM carboxy-methyl - NES Nethylsuccinimido     

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     

Ganglioside lactones:1H-NMR determination of the inner ester position of GD1b-ganglioside lactone naturally occurring in human brain or produced by chemical synthesis

The complete definition of the chemical structure of G_D1b-ganglioside (G_D1b) lactone isolated from human brain has been given by means of spectrometric and spectroscopic analyses. G_D1h lactone contains a single ester linkage involving the external sialic acid carboxyl group and the C-9 hydroxyl group of the internal sialic acid unit. A synthetic lactone of G_D1b prepared treating G_D1b with glacial acetic acid characterized in the same way showed an identical chemical structure.Abbreviations: Ganglioside nomenclature is according to Svennerholm [16] and the IUPAC-IUB Recommendations [17] G_M1 G_M1-ganglioside, II³NeuAc-GgOse₄Cer, Gal1-3GalNac1-4[NeuAc2-3]Gal1-4Glc1-1Cer - G_D1b G_D1b-ganglioside, II³(NeuAc)₂GgOse₄Cer, Gal1-3GalNAc1-4[NeuAc2-8NeuAc2-3]Gal1-4Glc1-1Cer - G_D1b lactone G_D1b-L, Gal1-3GalNAc1-4[NeuAc(1-9)2-8NeuAc2-3]Gal1-4Glc1-1Cer - Cer ceramide - FAB-MS fast atom bombardment-mass spectrometry - ¹H-NMR proteon nuclear magnetic resonance - 1D-NMR one dimensional NMR - 2D-COSY two dimensional correlated spectroscopy - DMSO-d₆ deuterated dimethylsulfoxide     

Lectin analysis of human immunoglobulin G N-glycan sialylation

The lectins Sambucus nigraagglutinin (SNA) and Ricinus communisagglutinin (RCA), specific for 2,6 linked sialylation, and terminal galactose respectively were used to study the occurrence, linkage and distribution of human immunoglobulin G (IgG) sialylation. SNA was shown to bind N-glycan 2,6-linked sialic acid only. Sialidase analysis confirmed that this is the dominant, if not exclusive linkage. Total IgG sialylation was estimated at 1.0[emsp4 ]g SA/mg IgG (or about 0.5 mole per mole) using a biochemical sialic acid assay. SNA displayed strong binding to the IgG Fab fragment in both its native and denatured state. In contrast, SNA failed to bind the IgG Fc fragment in its native form, but displayed strong binding after the Fc was denatured. This allowed the construction of quantitative assays capable of measuring both IgG Fab and Fc 2,6-sialylation without the need for enzymatic peptide digestion.     

Über das Wachstum der Pankreaszellen von Säugetieren als Monolayer Cultures

Zusammenfassung Glucose und Coffein erhöhen den Gehalt 4 und 7 Tage alter Zellen an immunologisch meßbarem Insulin (Ausgangsmaterial: Ratten- und Schweinepankreas). Morphologisch (Aldehydfuchsin, Pseudoisocyanin) sind bei coffeinbehandelten Zellen in der Intensität der Anfärbung und in der Granuladichte zeitabhängige Unterschiede zu beobachten, die an 4 Tage alten Zellen deutlicher als an 7 Tage alten zu erkennen sind. Coffein erhöht die Verfettung der Zellen. Weiter nimmt der Gehalt an sauren Mucopolysacchariden (Eisenbindungsreaktion) und an PAS-positivem Material zu. Diese Erhöhung läuft mit dem Anstieg des Insulingehaltes im Nährmedium parallel.

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Monolayer cultures of pancreatic tissueIII. Insulin release induced by coffein and glucose

Summary Glucose and caffeine increase the immunologically measurable insulin content of four and seven-day old cells (starting material: rats' or pigs' pancreas). In morphological examination (aldehyde fuchsin, pseudoisocyanin), time-dependent differences in the intensity of staining and granular density are observable in caffeine-treated cells; these phenomena are more distinct in four-day old cells than in seven-day old ones. Caffeine increases fatty degeneration of the cells. Furthermore, the content of acid mucopolysaccharides (iron-binding reaction) and of PAS positive material increases. This increase runs parallel with the rise in insulin content of the nutritive medium.

     

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.     

Variation in the karyotype of three cultivars of Narcissus tazetta L. (Amaryllidaceae)

Three cultivated varieties of Narcissus tazetta, Grand Soleil d'Or, Chinese Sacred Lily and Cypri, are triploid (2n = 3x+30) with the basic number 10. Grand Soleil d'Or has three homomorphic sets, each comprising 2 long submetacentrics, 4 long acrocentrics and 4 short acrocentrics. Karyotypes of the other two varieties are heteromorphic. Both possess one telocentric satellited chromosome. In addition, Cypri shows translocation between two chromosomes belonging to the seventh and eighth triplets. The number of secondary constrictions varies between 3 (Chinese Sacred Lily) and 4 (Grand Soleil d'Or and Cypri) which is also the number of nucleoli observed in the respective varieties.     

Development and parasitism in the genus Hemileia (uredinales)

Seventeen species ofHemileia were studied with emphasis on these characters: extent of colonization, specialization of parasitic mycelium, haustorial development, pathologic histology, soral morphology and sporulation capacity. An attempt was made to trace the varying degree of development and parasitism in this rust genus comprising of just over 40 reported species. It was noted that theGopalkrishnan's Subepidermal type more advanced than his Superstomal — B type.Fourth part of my thesis submitted to the University of Poona, India, accepted in 1968 for Ph.D. Not published.