Studies on the reductive amination of 3-deoxy-D-manno-octulosonic acid (Kdo).

Reductive amination of 3-deoxy-D-manno-octulosonic acid (Kdo) with allylamine (AIIN) or 2-(4-aminophenyl)ethylamine (APEA) yields epimer pairs of 2-N-allylamino and 2-N-[2-(4-aminophenyl)ethylamino]-2,3-dideoxy-D-glycero-D-galacto- and -2,3-dideoxy-D-glycero-D-talo-octonic acid. The yields were 50-60% due to reduction of Kdo to the respective polyols as side reaction products. Mass spectrometric analyses proved the amination derivatives to be the expected glycamines. Nuclear magnetic resonance (NMR) studies were performed on 2-N-allylamino-2,3-dideoxyoctonic acid which represents the chain terminus of allylaminated oligosaccharides derived from bacterial lipopolysaccharides (LPS) after acid hydrolysis and reductive allylamination.

Structure of the Acetobacter methanolicus MB 129 capsular polysaccharide, and of oligosaccharides resulting from degradation by bacteriophage Acm7.

The capsular polysaccharide of Acetobacter methanolicus MB 129 consists of D-Glc, D-Gal, L-Rha, and L-glyceric acid in the molar ratios 1:1:1:0.3. Periodate oxidation, methylation analysis, solvolysis with HF, and detailed 1H and 13C NMR analysis resulted in the structure of the repeating unit shown below. [formula: see text] Bacteriophage Acm7-associated end-alpha-L-rhamnopyranoside hydrolase depolymerizes the CPS even in the presence of the O-acyl group, to give the respective hexa-, nona-, and dodeca-saccharides.

Structure of the capsular polysaccharide and the O-side-chain of the lipopolysaccharide from Acetobacter methanolicus MB 70, and of oligosaccharides resulting from their degradation by the bacteriophage Acm6.

Acetobacter methanolicus MB 70 was shown to be related to the type strain of this species MB 58/4 (IMET 10945) having the same galactan-->2)-beta-D-Gal f-(1-->3)-beta-D-Gal p-(1-->as the capsular polysaccharide (CPS) and the O-side-chain of the lipopolysaccharide (LPS). Additionally, a glucan built up of the disaccharide repeating unit-->6)-alpha-D-Glc p-(1-->2)-alpha-D-Glc p-(1-->was identified in strain MB 70. In the CPS, the polymers were present in the ratio approximately 1:1, whereas the glucan preponderated in the LPS. Bacteriophage Acm6 specific to A. methanolicus MB 70 hydrolysed selectively the glucan component of both CPS and LPS. Structural elucidation of the resulting oligosaccharides led to the identification of the phage-associated depolymerase as an endo-alpha-(1-->6)-D-glucopyranoside hydrolase.

The chemical structure of bacterial endotoxin in relation to bioactivity.

Lipopolysaccharides (LPS) constitute the O-antigens and endotoxins of Gram-negative bacteria. Whereas both the polysaccharide and lipid portion of LPS contribute to the pathogenic potential of this class of bacteria, it is the lipid component (lipid A) which determines the endotoxic properties of LPS. The primary structure of lipid A of various bacterial origin has been elucidated and Escherichia coli lipid A has been chemically synthesized. The biological analysis of synthetic lipid A partial structures proved that the expression of endotoxic activity depends on a unique structural arrangement and conformation. Such analyses have furthermore provided insight into the determinants required for lipid A binding to and activation of human target cells. Present research efforts aim at the molecular characterization of the specificity, modulation and biomedical consequences of the interaction of lipid A with host cells.

Structure of the capsular polysaccharide and the O-side-chain of the lipopolysaccharide from Acetobacter methanolicus MB 58/4 (IMET 10945), and of oligosaccharides resulting from their degradation by the bacteriophage Acml.

The capsular polysaccharide (CPS) and the O-side-chain of the lipopolysaccharide (LPS) of Acetobacter methanolicus MB 58/4 (IMET 10945) have been shown to contain the same disaccharide repeating unit, namely, ----2)-beta-D-Galf-(1----3)-beta-D-Galp-(1----. Degradation of the CPS and the LPS with the bacteriophage Acml gave fragments built up of 1-5 repeating units; the octasaccharide preponderated. The phage-associated depolymerase proved to be a beta-D-galactofuranoside hydrolase.

[Effect of pepstatin A on Candida albicans infection in the mouse]. / Die Wirkung von Pepstatin A auf die Candida-albicans-Infektion der Maus.

The intravenous injection of 10(6) to 10(7) Candida albicans cells revealed to be a reliable model in mice produce infections of the kidney. Higher germ contents could be yielded in the kidney after the application of protease positive Candida-strains as compared to protease negative ones. Additionally, after the infection with protease positive strains a proteolytic activity could be found in the kidney homogenates in vitro on casein plates. The modulation of this Candida infection by pepstatin A, an inhibitor of extracellular yeast proteases in vitro, has also been studied in the same mice model in vivo. The growth rate of Candida albicans has been measured in the left kidney by counting the germ content as described by Haenel. Infections could be reduced after single doses of 120 micrograms pepstatin A contrary to 60 micrograms pepstatin A. The same was with three doses of 180 micrograms at 24 h intervals. This protease inhibitory effect could also be found in kidney homogenates in vitro on casein plates and lasted until 48 h post injection. On the basis of this positive effects on the Candida infection in mice pepstatin A should be considered as an adjuvans in the therapy of severe yeast infections.

[Slide test for immunofluorescent microscopy and immunoenzyme detection of cell-membrane antigens]. / Objektträgertest zur immunfluoreszenzmikroskopischen und enzymimmunologischen Erfassung von Zellmembranantigenen.

Poly-dimethyl-diallyl-ammoniumchloride - coated glass slides cause a strong adherence of living cells in consequence of electrostatic interactions. Immunological reactions of adherent cells - we used immunofluorescence and immunoperoxidase techniques to demonstrate membrane antigens - are not impaired. The slide test is suitable for routine quantitation of B and T cells in the peripheral blood and for the selection of hybridoma antibodies against cellular antigens. The slide test is time saving and needs minimal volumes of cells and antisera.

[Polysaccharide structure of cell wall preparations from the food protein yeast Candida spec. H]. / Polysaccharid-Strukturen von Zellwand-Präparaten aus der Futtereiweiss-Hefe Candida spec. H.

More than 27% of the cell wall are unstably bound components: Proteophosphomannan as a main polysaccharide of the cell wall, mannose and manno-oligosides which are bound to peptides or phosphate. 19% are glycosidically linked with a phosphate bridge or O-glycosidically (Ser/Thr) linked with the protein which is covalently bound to the cell wall. Besides mannose and glucose, manno-oligosides and gluco-oligosides are involved in this linkage. A preparation consisting of glucan and chitin remains after careful degradation. It contains (1,2)-, (1,3)- and (1,6)- linked glucose, (1, 2, 3)-, (1, 2, 6)- or (1, 3, 6)-glucose-branchpoints and (1,4)-linked N-acetylglucosamine.

[Structure of the cell wall polysaccharide in the food protein yeast Candida spec. H. VI. Isolation and structure of the glucans]. / Struktur der Zellwandpolysaccharide in der Futtereiweiss-Hefe Candida spec. H. VI. Isolierung und Strukturaufklärung der Glucane.

Alkali-soluble glucan was obtained from Candida spec. H by extraction with dilute sodium hydroxide. This minor component is quite heterogeneous in molecular size, has a highly branched structure and contains 19% beta (1,3)-, 24% beta (1,6)- and only 2% beta (1,2)-linkages. Other minor polysaccharide components have been isolated by acetic acid extraction. They have been identified as glycogen and linear beta(1,6)-glucan, respectively. The major component is a 1,2-, 1,3,-, 1,6-glucan branched in 1,2,3-, 1,3,6- and 1,2,6-positions, containing chitin. So we can confirm that different yeasts may differ in glucan structures which vary considerably both in the degree of branching and molecular size.

[Cell wall polysaccharide structure in the food protein yeast, Candida spec. H. II. Characterization of phosphate binding to the mannan-protein-phosphate complex and identification of phosphodiester-bound mono- oligosaccharides]. / Struktur der Zellwandpolysaccharide in der Futtereiweiss-Hefe Candida spec. H. II. Charakterisierung der Bindung des Phosphats am Mannan-Protein-Phosphate-Komplex und Identifizierung der als Phosphodiester gebundenen Mono- und Oligosaccharide.

The proteophosphomannan (PPM) obtained by extraction with citrate buffer, purification by the cetavlon method and gel filtration contains a high proportion of diesterified phosphate groups between the C-6 of a side chain mannose and another glycosidically bound carbohydrate. Mild acid hydrolysis cleaved the phosphate diester linkages to yield mono- and oligosaccharide fractions. Chemical analysis and 1H-NMR studies demonstrated the heterogeneity of the oligosaccharide fractions containing alpha(1,2)-, alpha(1,3)- and alpha(1,6)-linked manno-oligosaccharides. All repeated PPM preparations contained mono-, tri- and heptasaccharides as the phosphate bound main compounds. Galactose, arabinose, fucose, and rhamnose of the monosaccharide fraction and tetra-, penta- and hexasaccharides were not found in all preparations.

[Cell wall polysaccharide structure in the food protein yeast, Candida spec. H. I. Structure of alkali-stable mannoproteins]. / Struktur der Zellwandpolysaccharide in der Futter-eiweiss-Hefe Candida spec. H.

The isolated manno-protein contains about 80% mannose and 10% glucose. Methylation analysis established the highly branched nature of this polysaccharide and the presence of 1,2-, 1,3- and 1,6-linkages, as well as the linkages of the branch points. The research of the acetolysis fragments revealed that the molecule is composed on mannose and mannooligosaccharides with DP2 to DP12. These oligosaccharides are terminated in the nonreducing end by alpha(1,3)-mannose. Glucose was only found in the monosaccharide fraction corresponding to the nonsubstituted backbone and in the alpha(1,3)-disaccharide fraction (reducing and nonreducing end) of the acetolysis. A heptasaccharide fraction corresponding to the N-glycosidical linkage region between polysaccharide and protein parts of the glycoprotein had been isolated. 1H-NMR spectroscopy and chemical characterization made it probable that the unit with the first side chain, mannopentaose, is linked by di-N,N' -acetylchitobiose or by 4-0-beta-D-glucosyl-N-acetyl-D-glucosamine to the asparagine residue of the protein.

[Structure of the cell wall polysaccharide in the food protein yeast Candida spec H. III. Characterization of different phosphate bonds in the mannan-protein-phosphate complex]. / Struktur der Zellwandpolysaccharide in der Futtereiweiss-Hefe Candida spec. H. III. Charakterisierung unterschiedlicher Phosphatbindungen im Mannan-Protein-Phosphat-Komplex.

The 31P-NMR spectra of the proteophosphomannan (PPM) and also that of mildly hydrolyzed PPM demonstrated phosphomonoester (in both preparations), acid labile and acid stable phosphodiester linkage, and polyphosphate. Decreasing in size by pronase digestion, separation, purification and characterization of the high and low molecular phosphates by 31P-NMR spectroscopy and chemical analysis revealed the mannan protein is phosphorylated in the N-glycosidically linked carbohydrate parts and in the O-glycosidically linked oligosaccharides. Another phosphate serves as a bridge between the serine of the protein and mannose, mannobioses and mannotrioses and between the threonine and a lipophilic acylglycerid unit. The origin of the polyphosphates has been discussed.

[Structure of the cell wall polysaccharide in the food protein yeast Candida spec. H. IV. Structure of the alkali labile oligosaccharide in the mannan-protein-phosphate complex]. / Struktur der Zellwandpolysaccharide in der Futtereiweiss-Hefe Candida spec. H. IV. Struktur der alkalilabilen Oligosaccharide im Mannan-Protein-Phosphat-Komplex.

Mild alkaline degradation of the proteophosphomannan (PPM) under conditions that effect beta-elimination of the residues, O-glycosidically bound on serine and threonine, release mainly mannose, glucose, mannobioses and mannotrioses with the same structures as those obtained by acetolysis of the polysaccharide component of the PPM. Methylation analysis and paper chromatography demonstrated the structural heterogeneity of the tetra-, penta- and hexasaccharide fractions containing 1,2- and 1,6-linked and 1,2,6-branched manno-oligosaccharides. Methylation analysis and acetolysis, paper chromatography and analysis of the carbohydrate composition of the oligosaccharide fractions DP 7-12 and DP 15-19 demonstrated inner core region like structures containing 1,2- and 1,6-linked mannose, 1,2,6- and 1,3,6-branchpoints and N-acetyl-D-glucosamine.