Structural study on a phosphorylated mannotetraose obtained from the phosphomannan of Candida albicans NIH B-792 strain by acetolysis

A mixture of phosphorylated manno-oligosaccharides was isolated from the acid-stable domain of phosphomannan of Candida albicans NIH B-792 strain (serotype B) by acetolysis and was fractionated on a column of Bio-Gel P-2 equilibrated with 50 mM pyridine-CH3COOH buffer, pH 5.0. A monophosphorylated mannotetraose was isolated as the major constituent. Structural analyses of this phosphate-containing tetraose and its reduction product with NaBH4 by 1H, 13C, and two-dimensional homonuclear Hartmann-Hahn NMR spectroscopies, subsequently, gave results consistent with the structure described below (where Manp represents the mannopyranose unit): [formula: see text] It was unexpected that the major phosphorylated branch in the acid-stable domain of the parent phosphomannan of this C. albicans strain is a relatively short mannotetraosyl residue containing solely alpha-1,2-linked mannopyranose units, and a phosphate group as a 6-O-ester on the intermediary unit adjacent to the nonreducing terminal group. These findings indicate that the size of the major phosphorylated branch of this phosphomannan is the same as that of Saccharomyces cerevisiae.     

Molecular conformation of polysaccharides in solution. Changes in the optical rotation and in the elution pattern of gel filtration

Molecular conformation of some polysaccharides in aqueous solution in evidenced by changes in the optical rotation and in the elution pattern of gel filtration. The changes in the specific rotation against that in water are expressed as a molar conformational value [K]: ?495° for colominic acid in 1.0 N NaOH solution, and ?180° for hyaluronate (HA), +85° for corneal keratin sulfate, and +234° for amylose in 8 M urea solution. The gel filtration of amylose and HA dissolved in 8 M urea solution shows elution patterns distinctly different from those dissolved in water. The phenomena are attributable to a molecular conformational transition of polysaccharide molecules in aqueous solution.     

Sequential nuclear magnetic resonance assignment of beta-1,2-linked mannooligosaccharides isolated from the phosphomannan of the pathogenic yeast Candida albicans NIH B-792 strain.

The H-1 and H-2 signals of beta-1,2-linked mannooligosaccharides isolated from the phosphomannan of Candida albicans NIH B-792 strain by mild acid hydrolysis were assigned by a sequential NMR assignment method that combines two-dimensional 1H-1H correlated spectroscopy (COSY) and two-dimensional nuclear Overhauser enhancement and exchange spectroscopy (NOESY). The results indicated that the H-1 and H-2 of each beta-1,2-linked mannopyranose unit show largely different signals compared with those of the alpha-linked ones and that the correlation between linkages and signals could not be explained by a conventional additivity rule. Furthermore, a regular proportional downfield shift of the H-1 signal was observed in the order of the mannose unit from the reducing terminal except those of the reducing and nonreducing terminal positions. Although the 1H NMR spectra of these oligosaccharides were complicated due to the presence of a large portion of the beta-anomer from the reducing terminal mannose unit, reduction of the oligosaccharides with NaBH4 to the corresponding alcohols gave simple and more readily interpretable 1H NMR spectra. Unexpectedly, however, a shift of H-1 signals by this reduction occurred not only on the second mannose unit but also on the third and fourth mannose units from the modified reducing terminal group of each oligosaccharide alcohol. This result indicates that the reducing terminal mannose unit is able to affect up to the fourth mannose unit from the reducing terminal. The presence of a long-distance interresidue NOE also suggests that the beta-1,2-linked mannooligosaccharides have a compactly folded conformation in solution.     

Expression of chimeric ras protein with OmpF signal peptide in Escherichia coli: localization of OmpF fusion protein in the inner membrane

Summary The ras gene was fused with the DNA sequence of OmpF signal peptide or with the DNA sequence of OmpF signal peptide plus the amino terminal portion of the OmpF gene. They were placed in plasmids together with the bacteriophage P _L promoter. These plasmids were introduced into Escherichia coli strain K-12 and the OmpF signal peptide fusion proteins were expressed. These fusion proteins were idetified as 29.0 and 30.0 kDa proteins. However, processed products of these proteins were not found in the The fusion proteins were localized mostly in the cytoplasm and the inner membrane, but none of them was secreted into the periplasmic space. On the other hand, the ras protein alone was found in the cytoplasm and not in the inner membrane. Viable counts of E. coli harbouring these plasmids decreased when these fused proteins were induced. Induction of the ras protein alone did not harm cells. These observations suggest that insertion of the heterologous proteins into the inner membrane may cause the bactericidal effect. Offprint requests to: A. Kaji     

Metabolic diversification of nitrogen‐containing metabolites by the expression of a heterologous lysine decarboxylase gene in Arabidopsis

Lysine decarboxylase converts l ‐lysine to cadaverine as a branching point for the biosynthesis of plant Lys‐derived alkaloids. Although cadaverine contributes towards the biosynthesis of Lys‐derived alkaloids, its catabolism, including metabolic intermediates and the enzymes involved, is not known. Here, we generated transgenic Arabidopsis lines by expressing an exogenous lysine/ornithine decarboxylase gene from Lupinus angustifolius (La‐L/ODC) and identified cadaverine‐derived metabolites as the products of the emerged biosynthetic pathway. Through untargeted metabolic profiling, we observed the upregulation of polyamine metabolism, phenylpropanoid biosynthesis and the biosynthesis of several Lys‐derived alkaloids in the transgenic lines. Moreover, we found several cadaverine‐derived metabolites specifically detected in the transgenic lines compared with the non‐transformed control. Among these, three specific metabolites were identified and confirmed as 5‐aminopentanal, 5‐aminopentanoate and δ‐valerolactam. Cadaverine catabolism in a representative transgenic line (DC29) was traced by feeding stable isotope‐labeled [α‐¹⁵N]‐ or [ε‐¹⁵N]‐l ‐lysine. Our results show similar ¹⁵N incorporation ratios from both isotopomers for the specific metabolite features identified, indicating that these metabolites were synthesized via the symmetric structure of cadaverine. We propose biosynthetic pathways for the metabolites on the basis of metabolite chemistry and enzymes known or identified through catalyzing specific biochemical reactions in this study. Our study shows that this pool of enzymes with promiscuous activities is the driving force for metabolite diversification in plants. Thus, this study not only provides valuable information for understanding the catabolic mechanism of cadaverine but also demonstrates that cadaverine accumulation is one of the factors to expand plant chemodiversity, which may lead to the emergence of Lys‐derived alkaloid biosynthesis.     

The quality of sleep and factors associated with poor sleep in Japanese graduate students

Sleep and Biological Rhythms - We conducted a cross-sectional mail questionnaire survey at 31 graduate schools on 12 university campuses in Kyoto, Japan to assess the sleep quality of the graduate...