Developmentally regulated O-acetylated sialoglycans in the central nervous system revealed by a new monoclonal antibody 493D4 recognizing a wide range of O-acetylated glycoconjugates

We have previously detected an alkali-labile and developmentally regulated antigen in rat embryonic cerebral cortex, which may be 9-O-acetylsialylated GT3 ganglioside (Hirabayashi Y, Hirota M, Suzuki Y, Matsumoto M, Obata K, Ando S (1989) Neurosci Lett 106:193-98). In this study we established a mouse monoclonal antibody, 493D4, that recognizes 9-O-acetyl GT3 ganglioside, but not non-O-acetyl gangliosides. This antibody also reacted with 9-O-acetyl GD3 to a much lesser extent. By using this antibody, we found that O-acetyl GT3 as well as O-acetyl GD3 were expressed strongly in fetal murine cerebral cortex and decreased to an undetectable level after birth. With the assistance of TLC-immunostaining using 493D4 together with Q-Sepharose column chromatography, O-acetyl gangliosides of bovine brain were purified and the structural analysis showed the presence of O-acetyl GD3, O-acetyl LD1, O-acetyl GD2 and O-acetyl GD1b in the adult brain as extremely minor components. Interestingly, the antibody 493D4 could detect O-acetyl sialoglycoproteins in rat brain tissues. One of the major immunoreactive proteins was shown to be synaptophysin, an integral membrane protein specifically present in synaptic vesicles. This monoclonal antibody was therefore useful for sensitive detection of both O-acetylated gangliosides and glycoproteins with O-acetylated sialic acids. This revised version was published online in November 2006 with corrections to the Cover Date.     

Letter to the editor Synthesis of 16-fluoromethylene-prostaglandin F2α

Prostaglandin (PG) E₂, thromboxane (TX) B₂ and the stable breakdown product of prostacyclin, 6-oxo-PGF_1α are present in carrageenin-induced inflammatory exudates. Carrageenin-impregnated polyester sponges were implanted subcutaneously in rats and inflammatory exudates were collected 4–192 h after implantation. The concentrations of cyclo-oxygenase products in sponge fluids was measured by radioimmunoassay after extraction and purification. All three products were detectable after 4 h and reached a peak at 12–24 h. Mean TXB₂ concentrations reached 74 ng/ml at 12 h but decreased to less than 10 ng/ml after 24 h. PGE₂ concentrations were 65 ng/ml at 24 h, after which there was no significant increase and then dropped to about 20 ng/ml between 96 and 192 h. 6-oxo-PGF_1α reached a concentration of 33 ng/ml at 24 h which did not change significantly until levels fell to less than 10 ng/ml between 96 and 192 h. The presence of PGE₂, TXB₂ and 6-oxo-PGF_1α was confirmed by gas-liquid chromatography and mass spectrometry. Total leukocyte numbers increased steadily and were at their highest (116.0 × 10⁶ cells/ml) at 192 h. These results suggest that thromboxanes and prostacyclin, as well as PGE₂, contribute to the acute inflammatory response.     

Structures and physiological roles of 13 integral lipids of bovine heart cytochrome c oxidase

All 13 lipids, including two cardiolipins, one phosphatidylcholine, three phosphatidylethanolamines, four phosphatidylglycerols and three triglycerides, were identified in a crystalline bovine heart cytochrome c oxidase (CcO) preparation. The chain lengths and unsaturated bond positions of the fatty acid moieties determined by mass spectrometry suggest that each lipid head group identifies its specific binding site within CcOs. The X-ray structure demonstrates that the flexibility of the fatty acid tails facilitates their effective space-filling functions and that the four phospholipids stabilize the CcO dimer. Binding of dicyclohexylcarbodiimide to the O(2) transfer pathway of CcO causes two palmitate tails of phosphatidylglycerols to block the pathway, suggesting that the palmitates control the O(2) transfer process.The phosphatidylglycerol with vaccenate (cis-Delta(11)-octadecenoate) was found in CcOs of bovine and Paracoccus denitrificans, the ancestor of mitochondrion, indicating that the vaccenate is conserved in bovine CcO in spite of the abundance of oleate (cis-Delta(9)-octadecenoate). The X-ray structure indicates that the protein moiety selects cis-vaccenate near the O(2) transfer pathway against trans-vaccenate. These results suggest that vaccenate plays a critical role in the O(2) transfer mechanism.     

Determination of 3-hydroxyisovalerylcarnitine and other acylcarnitine levels using liquid chromatography-tandem mass spectrometry in serum and urine of a patient with multiple carboxylase deficiency

Due to its increased concentration in blood, 3-hydroxyisovalerylcarnitine (C5OH-I) is an important indicator for the diagnosis of organic acidemias in newborns. However, C5OH-I has not been used as a standard in tandem mass spectrometric (MS/MS) assays because its isolation is difficult. We developed a new synthesis of C5OH-I and investigated its behavior by MS/MS. A method using the multiple reaction monitoring (MRM) mode of MS/MS with HPLC was developed which provides high accuracy, precision and reproducibility. Acylcarnitine profiles in the serum and urine of a patient with multiple carboxylase deficiency (MCD) showed increased levels compared to a healthy patient.     

Synthesis of new sugar derivatives and evaluation of their antibacterial activities against Mycobacterium tuberculosis

A series of sugar derivatives (1–13) were synthesized and evaluated for antibacterial activity against Mycobacterium tuberculosis (MTB), especially multi-drug resistant (MDR) MTB, and the structure–activity relationships of these compounds were studied. The results showed that the compound OCT313 (2-acetamido-2deoxy-β-d-glucopyranosyl N,N-dimethyldithiocarbamate) (4) exhibited significant in vitro bactericidal activity, and that the dithiocarbamate group at C-1 position of the glucopyranoside ring was requisite for the antibacterial activity.     

Identification of a novel DNA element that promotes cell-to-cell transformation in Escherichia coli

Recently, we discovered a novel phenomenon, "cell-to-cell transformation" by which non-conjugative plasmids are transmitted horizontally in co-cultures of Escherichia coli F(-) strains. In this study, we aimed to identify the DNA element responsible for the high cell-to-cell transformability of pHSG299. By transplanting pHSG299 DNA fragments into pHSG399, a plasmid showing low transformability, we discovered that a specific 88 bp fragment of pHSG299 significantly promoted pHSG399 transformability. Although several short motif-like repetitive sequences (6-10 bp) were present in the 88 bp sequence, no known DNA motifs were recognized, suggesting that this 88 bp sequence (cell-to-cell transformation promoting sequence, CTPS; Accession number: AB634455) is a novel DNA element.     

Analyses of N-linked oligosaccharides using a two-dimensional mapping technique

We propose a two-dimensional sugar map method for the simple, reproducible, and sensitive analysis of the structures of N-linked oligosaccharides. The structure of an unknown oligosaccharide can be characterized from its position on the map. The data base for the sugar map is prepared by the use of 113 kinds of standard oligosaccharides, 58 of whose structures have been confirmed by 1H NMR spectroscopy. The present method involves six steps, (i) preparation of oligosaccharides from glycopeptides by N-oligosaccharide glycopeptidase (almond) digestion, (ii) derivatization of the reducing ends of oligosaccharides with a fluorescent reagent, 2-amino-pyridine, by using sodium cyanoborohydride, (iii) separation of oligosaccharide derivatives by high-performance liquid chromatography with an ODS-silica column, (iv) analysis of the size of each separated oligosaccharide on an amide-silica column, (v) plotting of the elution position of a sample on the two-dimensional sugar map obtained for the standard oligosaccharides, and (vi) structural analysis of the oligosaccharides by a combination of sequential exoglycosidase digestion and the steps (iii-v). The present method was applied to the identification of the structures of oligosaccharides in hen ovalbumin. It was found that two unusual oligosaccharides that have not yet been reported exist in ovalbumin.     

Sensitive determination of xylenes in whole blood by capillary gas chromatography with cryogenic trapping

A new and sensitive method for measurement of o-, m- and p-xylenes in human whole blood by capillary gas chromatography (GC) with cryogenic trapping is presented. After heating 0.5 ml of whole blood and 0.5 ml of distilled water containing the xylenes and aniline (internal standard, I.S.) in a 4.0-ml vial at 100°C for 30 min, 2 ml of the headspace vapor was drawn into a glass syringe. All vapor was introduced through the GC port into an AT-Wax middle-bore capillary column in the splitless mode at an oven temperature of 5°C to trap the entire analytes, and the oven temperature was then programmed up to 180°C. The present conditions gave sharp peaks for xylenes and aniline (I.S.), and low background noises for whole blood samples; the peaks of p- and m-xylenes showed about 90% separation with the AT-Wax column. As much as 41.0–46.3% of xylenes, which had been spiked to whole blood could be recovered. The calibration curves showed linearity in the range of 0.1–0.5 μg/0.5 ml of whole blood. The detection limit was estimated to be about 10 ng/0.5 ml. The coefficients of intra-day and inter-day variations for xylenes were not greater than 9.38%. The data for actual detection of xylenes in post-mortem blood of self-ignition suicide cases by the present method were also presented.