Fritz Zilliken

Antifungal activity of soybean and chickpea isoflavones and their reduced derivatives

Abstract The fungicidal activity of the isoflavones from soybean ( Glycine max ) and chickpea ( Cicer arietinum ) has been studied on three food and forage contaminating fungi, Aspergillus ochraceus , Penicillium digitatum and Fusarium culmorum . The reduced derivatives of the corresponding isoflavones—the isoflavanones and the isoflavans—were also included in the investigation. For the first time in a comparative study it is shown that isoflavones and isoflavanones are variable in their activity whereas the isoflavans are moderately active inhibitors of fungal growth.

Uptake and Distribution of Orally Applied N-Acetyl-(14C)neuraminosyl-lactose and N-Acetyl-(14C)neuraminic Acid in the Organs of Newborn Rats

N-acetyl-(14C)neuraminosyl-(alpha,2 leads to 3)lactose enzymatically prepared of CMP-NeuNAc and lactose by a particulate enzyme fraction from lactating rat mammary gland was applied orally to newborn rats and examined for uptake and distribution in relation to those of free N-acetyl-(14C)neuraminic acid. The neonates were allowed to stay with their mother before and during the incubation time up to 6 h. Within this time 70% of the given dose was excreted while 30% was retained in the body. (14C)NeuNAc-lactose activity appeared 1.5 h after application in blood, urine, and tissues and attained maximum values after 3 and 6 h, respectively. The highest uptake occurred in liver, spleen, and brain. The absorption of the trisaccharide was delayed by 30 min compared with free (14C)NeuNAc. The time courses of the curves show a slower but higher accumulation in the tissues suggesting a better utilization of the (14C)NeuNAc from (14C)NeuNAc-lactose or pecularities in the absorption of the trisaccharide by the organs.

Minor constitutents of human milk IV: Analysis of the branched chain fatty acids

Abstract After catalytic hydrogenation of the unsaturated fatty acids, the branched chain fatty acids of human milk fat were enriched 100–500-fold by urea fractionation. The fatty acid (FA) mixture obtained was qualitatively and quantitatively analysed by a combination of gas chromatograph-mass spectrometer (LKB 9000) using packed columns, thin film and S.C.O.T. capillary columns. More than 50 single and multi branched acids could be identified. From the distribution of the individual branching points, it is concluded, that two major pathways exist for the biosynthesis of these acids: either by chain elongation of the branched CoA-esters (such as isobutyryl-CoA, α- and β-methylbutyryl-CoA) or by methylation of the double bond of monoenoic acids. The quantitative distribution of the branched acids strongly resembles that found in many micro-organisms. Hence it is assumed, that a large number of these acids is produced in - or absorbed from - the intestinal tract.

A Newly Discovered Sialidase from Gardnerella Vaginalis

A sialidase (neuraminidase, acylneuraminosyl hydrolase, EC 3.2.1.18) has been discovered and isolated from Gardnerella vaginalis (ex. Haemophilus vaginalis), a possibly pathogenic inhabitant of the female genital tract. Bacteria were grown in peptone-yeast-extract medium with 2.0 mM N-acetylmannosamine as enzyme inductor under CO2 atmosphere. Sialidase activity was found in the bacterial sediment and in the culture medium. The enzyme was liberated from the cells by ultrasonic treatment. Purification was performed by 60-80% ammonium sulfate precipitation and by column chromatography on Sepharose CL-6B and Sephadex G 200. The enzyme revealed a molecular weight in the range of Mr 75 000 and a pH optimum at 5.5. Among the different types of NeuAc-containing glycoconjugates, the enzyme exhibits its highest activities towards the globular glycoproteins alpha 1-acid glycoprotein and fetuin. Taking their cleavage rate as 100, it is around 55 for II3NeuAc-Lac, 45 for bovine submaxillary mucin, 35 for II6NeuAc-Lac and IV3, III6NeuAc2-LcOse4. The rates for III8,II3NeuAc2-Lac, gangliosides and colominic acid are below 20. Due to its specificity pattern, the enzyme may play a role in the pathogenic process of G. vaginalis infections.

Receptors of neurotransmitters-II: Sialic acid metabolism and the serotonin induced contraction of smooth muscle

Abstract A number of different N -acylneuraminic acids, gangliosides, neuraminidases and synthetic inhibitors of sialic acid biosynthesis have been tested on the serotonin induced contraction of the rat stomach fundus. Gangliosides and N -acylneuraminic acids were found to increase sensitivity and maximal contraction height of the fundus preparation. In contrast the inhibitors decreased the rate of contraction. The serotonin receptor and its complex with serotonin were broken down upon incubation with neuraminidases. There was found a certain relationship between the inhibitory action of sialic acid biosynthesis in the cell-free system and the regeneration of serotonin receptors in the muscle preparation.

The absorption and fluorescence of isoflavones and the effect of shift reagents

The absorption and fluorescence maxima of 20 isoflavones have been determined in methanol solution and the effect of addition of water, 50% sulfuric acid, aluminium trichloride, borax, sodium acetate, ammonia and sodium hydroxide has been studied. The following findings may be useful in the structure elucidation of naturally occurring isoflavones: (a) 5-Hydroxyisoflavones have band I absorption maxima around 335 nm. 6-hydroxyisoflavones between 310 and 330 nm, and others below 310 nm. (b) Addition of water produces practically no shift in the absorption spectra, but - unlike other hydroxyisoflavones - can give distinctly longwave shifted new fluorescence bands with 7-hydroxyisoflavones. (c) Addition of sodium acetate gives rise to anion absorption of 7-hydroxyisoflavones and to partial anion absorption of 6-hydroxyisoflavones; the spectral maxima of 5-hydroxyisoflavones remain practically unchanged, (d) Ammonia gives rise to anion absorption of both 6- and 7-hydroxyisoflavones, but not of the 5-hydroxy isomers, (e) Sodium borate is a useful reagent to identify 6,7-dihydroxyisoflavones by virtue of its ability to form a chelate complex with an absorption maximum that is different from the anion absorption, (f) Aluminium trichloride forms complexes with both 5-hydroxy- and 6,7-dihydroxyisoflavones with distinct absorption maxima, (g) 5,7-D ihydroxyisoflavones may be recognized by addition of ammonia, which does not result in a longwave shift, but rather in an intensification of the longwave absorption band, (h) 6-H ydroxyisoflavones can be differentiated from the 7-hydroxy isomers by their longwave shifts (4 0-60 nm) following addition of ammonia. The respective shifts of the 7-hydroxy isomers are smaller, (i) 5-H ydroxyisoflavones are practically non-fluorescent. while others have fairly strong fluorescences, (j) The absorption and fluorescence maxima of isoflavones give unique combinations which may be useful in their identification, (k) Addition of aluminium chloride makes the non-fluorescent 5-hydroxyisoflavones fluorescent. (1) As in the case of absorption, 6,7-dihydroxyisoflavones form complexes with borate possessing fluorescence bands with maxima different from those of the anion bands

Comparison of enzyme activities in Dicrocoelium dendriticum, bovine liver, and Fasciola hepatica.

SummaryEnzymes involved in glycolysis, tricarboxylic acid cycle and pentose shunt were determined in the lancet fluke, Dicrocoelium dendriticum. The results have been compared with those obtained in host liver (bovine) and in the liver fluke, Fasciola hepatica. Enzyme activities of Dicrocoelium dendriticum show a greater similarity to those of host liver. It is assumed that fermentation of glucose—in contrary to Fasciola hepatica—is mainly leading to lactate.ZusammenfassungIm Lanzettegel, Dicrocoelium dendriticum, wurden Enzyme des Glucoseabbaues, des Pentose-Phosphatcyclus und des Citratcyclus bestimmt und mit denen der Wirtsleber (Rind) und des Leberegels, Fasciola hepatica, verglichen. Die gemessenen Enzymaktivitäten bei Dicrocoelium dendriticum gleichen mehr denen der Wirtsleber. Es zeigt sich, daß bei Dicrocoelium dendriticum der Glucoseabbau im Gegensatz zu Fasciola hepatica bis zum Lactat wahrscheinlich ist.

Massenspektren synthetischer methylen- und nicht-methylen-unterbrochener cis, cis-Oktadecadiensäure-methylester als Tetra-O-trimethylsilyläther / Mass Spectra of Synthetic Methylene- and Non-Methylene-Interrupted cis-cis-Methyl-Octadecadienoates as Their Tetra-O-Trimethylsilylether

The 20-eV-Mass spectra of eight methylene- and eight non-methylene-interrupted cis,cis-methyl-octadecadienoates as their tetra-O-trimethylsilyl derivatives are given. The fragmentation pattern dependent on the original position of double bonds and number of methylene-groups between the two double bonds is discussed.

Isolation, Purification, and Properties of Neuraminidase from Propionibacterium acnes

Neuraminidase activity was discovered in 32 of 38 strains of Propionibacterium acnes. Enzyme production was studied in yeast extract bouillon of different pH containing various amounts of human milk as neuraminidase inductor. Enzyme activity was found in the bacterial sediments as well as in the culture filtrates. Since neither ultrasonic treatment nor lysozyme incubation of bacterial sediments did release reasonable amounts of enzyme, culture filtrates were used for enzyme preparation. Neuraminidase was isolated by 40% ammonium sulfate precipitation, dialysis, concentration and repeated gel chromatography on Sephadex G-100. The enzyme posesses a molecular weight of about 33 000 and a pH-optimum around 5.0. The Michaelis constants are 1.8 x 10(-3) M for alpha 2 leads to 3 linked N-acetylneuraminic acid (NeuAc) in II3NeuAc-Lac, 3.7 x 10(-3) M for the alpha 2 leads to 6 linkage in II6NeuAc-Lac, and 2.1 x 10(-3) M for the alpha 2 leads to 8 linkage of II3 (comes from 2 alpha NeuAc8)2-Lac, respectively. Among the different groups of naturally occurring NeuAc-containing substrates, i.e. oligosaccharides, glycolipids and glycoproteins, the enzyme exhibits its highest activity towards low molecular weight oligosaccharides. Activity is considerably lower on glycoproteins. Glycolipids (gangliosides) are only little attacked under conditions used in the test. However, there is no remarkable specificity towards one of the different linkage types of N-acetylneuraminic acid. In general, the enzyme reveals a specificity pattern similar to that found in other bacteria of low pathogenicity towards man.

Mass spectrometric sequence analysis of complex oligosaccharides. Comparison of the permethyl- and pertrimethylsilyl-derivatives of lacto-N-tetraose.

Complex oligosaccharides bound either to ceramides or to protein chains are integral components of cell surfaces [l-3] . Evidence has been presented by several laboratories that these oligosaccharide chains strongly influence the ‘social behaviour’ of cells like cell contact, contact inhibition of growth and movement, surface charge, immunological changes, antigenicity, agglutinability and viral and tumorigenic transformation [2-41 . It is obvious that there exists an increasing demand for structural elucidation of these carbohydrate chains. Among the tedious and material consuming methods for structural characterization like consecutive enzymatic degradation [S] periodate degration [6] and permethylation [7,8] mass spectrometry has proved to be a most powerful and elegant tool for microscale preparations. Mass spectrometric data have been presented for permethyl [9, lo] pertrimethylsilyl [l l] peracetyl [ 121 and cyclic boronic acid derivatives [ 13151 of monoand oligosaccharides as well as glycolipids. The data concerning complex oligosaccharides are still sparse and there exists still some controversy about optimal derivatization methods [lo] . In order to shed some light on this question, the mass spectra of /3methyl-dodeca-0-methyl-lacto-N-tetraose and trimethylsilyl-LNT are discussed in the following paper. Butylboronic acid leading to a mixture of derivatives was not included.