Heterogeneity of urinary oligosaccharides from mannosidosis: Mass spectrometric analysis of permethylated Man9, Man8, and Man7 derivatives

Three oligosaccharides isolated from the urine of a patient suffering from mannosidosis, a Man₉GlcNAc, a Man₈GlcNAc, and a Man₇GlcNAc, were analyzed by electron impact mass spectrometry at 20 eV after reduction with NaB²H₄ and permethylation. Molecular ions were observed at $\text{m}\text{e}$ 2144, 1940, and 1736, respectively. In the high-mass range very intense ions were found at M-45. The mass spectrum of the homogenous decasaccharide Man₉GlcNAc_1D contains ions that could be attributed to specific parts of each of the three antennae of the molecule. Thus characteristic key ions were recognized. With the aid of these key ions the spectra of the nona- and octasaccharide mixtures could be evaluated in a qualitative and a semiquantitative way. In the nonasaccharide all three possible isomers that can be produced by cleavage of one of the three terminal α-mannoses are present, although in differing amounts. However, only five of the six possible isomers of the octasaccharide could be detected.     

Structure determination of the glycans of human-serum alpha 1-antichymotrypsin using 1H-NMR spectroscopy and deglycosylation by N-glycanase

alpha 1-Antichymotrypsin purified from normal human serum was separated by affinity chromatography into th ree microheterogeneous forms on a concanavalin-A-Sepharose column: a pass-through (peak 1), a retarded (peak 2) and a bound form (peaks 3 + 4). For each form the asparagine-linked carbohydrate chains were liberated as oligosaccharides by hydrazinolysis, submitted to reduction with NaBH4 after re-N-acetylation and further separated by affinity chromatography on a concanavalin-A-Sepharose column. The complete primary structure of the glycans was determined by high-resolution 1H-NMR spectroscopy. The results indicated the presence of disialyl diantennary and of trisialyl triantennary type glycanic structures, the latter being accompanied by traces of disialylated triantennary oligosaccharide. The N-glycanase was used for the deglycosylation of the unfractionated alpha 1-antichymotrypsin; the successive removal of the N-linked complex-type oligosaccharide side chains of alpha 1-antichymotrypsin was studied in the presence of detergents. From these experiments it is concluded that alpha 1-antichymotrypsin carries four oligosaccharide side chains. Moreover our results show that the peak 1 contains four triantennary glycans, the peak 2 three triantennary and one diantennary glycans while the bound peaks 3 + 4 possess, on average, about one triantennary and three diantennary glycans per molecule. Since we showed that the peak 4 contains mostly diantennary glycans, it can be deduced that in peak 3 there are molecules carrying two triantennary and two diantennary glycans and others carrying one triantennary and three diantennary glycans.     

Characterization of Le(x), Le(y) and A Le(y) antigen determinants in KDN-containing O-linked glycan chains from Pleurodeles waltlii jelly coat eggs.

Novel acidic oligosaccharides were isolated in large amounts by reductive alkaline treatment of the jelly coat of Pleurodeles waltlii (Michah) eggs. The oligosaccharides were found to contain the newly described KDN as acidic monosaccharide and possess either the Le(x), Le(y) and A Le(y) antigenic determinants. Occurrence of Le(x) and Le(y) determinants previously recognized as tumor-associated antigen (TAA) demonstrates that mucins of lower animals may represent a rich and easily available source for preparing TAA. Moreover, it reinforces the hypothesis according to which TAA are evolution markers.     

1H-NMR spectroscopy of manno-oligosaccharides of the beta-1,2-linked series released from the phosphopeptidomannan of Candida albicans VW-32 (serotype A).

Manno-oligosaccharides (DP 2 to greater than 15) were released by mild acid hydrolysis from the phosphopeptidomannan of a Candida albicans strain of A serotype (VW-32). Manno-oligosaccharides ranging from biose to heptaose were obtained in appreciable amount. Structural investigation of these oligosaccharides showed them to be of the beta-1,2-linked series. The occurrence of such compounds has already been reported in other strains of Candida albicans. We here report the assignment of the structural reporter groups of each of them, and general rules applicable for the 1H-NMR spectrum analysis of linear manno-oligosaccharide of general structure: Man(beta 1-2) [Man(beta 1-2)]nMan     

Photosynthetic apparatus of chilling-sensitive plants IX. The involvement of α-tocopherol in the electron transport chain and the anti-oxidizing system in chloroplasts of tomato leaves

1. The role of tocopherols in tomato chloroplasts from fresh, cold and dark-stored as well as stored and illuminated leaves was studied.2. The cold and dark storage of leaves results in a loss of chloroplast α- and γ-tocopherols of about 30–40% accompanied by an increase in chloroplast δ-tocopherol of about 40%. On illumination of stored leaves, an elevation of α- and γ-tocopherol level to about 110 and 95% of the control, respectively, occurs, whilst δ-tocopherol content is not affected.3. Experiments performed with 2,2-diphenyl-1-picrylhydrazyl-treated chloroplasts show that only about 70% of total α-tocopherol is functionally active in the electron transport of Photosystem II between the diphenyl-carbazide (DPC) donation site and the inhibition site of DBMIB.4. A small amount of α-tocopherol quinone (about 10% of α-tocopherol content) is found in chloroplasts from fresh, fresh and illuminated as well as cold and dark-stored tomato leaves, whereas the illumination of the latter increases the chloroplast α-tocopherol quinone content 3-fold. Moreover, following the illumination of chloroplasts from cold and dark-stored as well as stored and illuminated leaves, the oxidation of exogenous α-tocopherol to α-tocopherol quinone is 2-fold faster then in chloroplasts from fresh leaves.5. The primary product (‘α-tocopheroxide’) formed during the α-tocopherol oxidation by illuminated chloroplasts was identified as 8a-hydroxy-α-tocopheron.6. Exogenous α-tocopherol inhibits the lipid photoperoxidation by about 40–50% in chloroplasts from all three kinds of tomato leaf.7. The results seem to suggest that chloroplast α-tocopherol is involved in both electron transport of PS II and antioxidizing system of chloroplasts.     

Endothelial Barrier Antigenimmunoreactivity is Conversely Associated with Blood-Brain Barrier Dysfunction after Embolic Stroke in Rats

While the concept of the Neurovascular Unit (NVU) is increasingly considered for exploring mechanisms of tissue damage in ischemic stroke, immunohistochemical analyses are of interest to specifically visualize constituents like the endothelium. Changes in immunoreactivity have also been discussed to reflect functional aspects, e.g., the integrity of the blood-brain barrier (BBB). This study aimed to characterize the endothelial barrier antigen (EBA) as addressed by the antibody SMI-71 in a rat model of embolic stroke, considering FITC-albumin as BBB leakage marker and serum levels of BBB-associated matrix metalloproteinases (MMPs) to explore its functional significance. Five and 25 h after ischemia onset, regions with decreased BBB integrity exhibited a reduction in number and area of EBA-immunopositive vessels, while the stained area per vessel was not affected. Surprisingly, EBA content of remaining vessels tended to be increased in areas of BBB dysfunction. Analyses addressing this interrelation resulted in a significant and inverse correlation between the vessels’ EBA content and degree of BBB permeability. In conclusion, these data provide evidence for a functional relationship between EBA-immunoreactivity and BBB dysfunction in experimental ischemic stroke. Further studies are required to explore the underlying mechanisms of altered EBA-immunoreactivity, which might help to identify novel neuroprotective strategies.Key words: ischemic stroke, embolic model, EBA, blood-brain barrier, matrix metalloproteinase     

N,N-Diisopropyl-O-Methyl-O-4-Nitrophenyl Phosphoramidite. New Flexible Phosphitylating Reagent in the Synthesis of Thio- and Seleno- Oligonucleotides

Abstract

Internucleoside coupling by the title reagent, illustrated by synthesis of dinucleoside and trinucleoside thiophosphates and selenophosphates is accomplished by a new strategy involving the displacement of 4-nitrophenoxy group. Sodium 4-nitrophenolate formed in situ facilitates the oxidation of the intermediate pIII structures and acts as a demethylating reagent.     

Frizzled-4 is required for normal bone acquisition despite compensation by Frizzled-8

The activation of the Wnt/β-catenin signaling pathway is critical for skeletal development but surprisingly little is known about the requirements for the specific frizzled (Fzd) receptors that recognize Wnt ligands. To define the contributions of individual Fzd proteins to osteoblast function, we profiled the expression of all 10 mammalian receptors during calvarial osteoblast differentiation. Expression of Fzd4 was highly upregulated during in vitro differentiation and therefore targeted for further study. Mice lacking Fzd4 in mature osteoblasts had normal cortical bone structure but reduced cortical tissue mineral density and also exhibited an impairment in the femoral trabecular bone acquisition that was secondary to a defect in the mineralization process. Consistent with this observation, matrix mineralization, markers of osteoblastic differentiation, and the ability of Wnt3a to stimulate the accumulation of β-catenin were reduced in cultures of calvarial osteoblasts deficient for Fzd4. Interestingly, Fzd4-deficient osteoblasts exhibited an increase in the expression of Fzd8 both in vitro and in vivo, which suggests that the two receptors may exhibit overlapping functions. Indeed, ablating a single Fzd8 allele in osteoblast-specific Fzd4 mutants produced a more severe effect on bone acquisition. Taken together, our data indicate that Fzd4 is required for normal bone development and mineralization despite compensation from Fzd8.